Compositions and methods for increasing erythropoietin (epo) production

ABSTRACT

The invention relates to double-stranded ribonucleic acid (dsRNA) compositions targeting one or more EGLN genes, EGLN1, EGLN2 and/or EGLN3 and methods of using such dsRNA compositions to inhibit expression of these genes.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority of Application Ser. No. 61/493,651filed on Jun. 6, 2011 and Application Ser. No. 61/421,727 filed on Dec.10, 2010 each of which are incorporated by reference in their entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made, in part, with government support under ContractNumber NIH CA068490 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

REFERENCE TO SEQUENCE LISTING

The present application is being filed along with a Sequence Listing inelectronic format. The Sequence Listing is provided as a file entitledALN144WO_SEQLST_final.txt created on Nov. 22, 2011 which is 632,824bytes in size. The information in electronic format of the sequencelisting is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The invention relates to the specific inhibition of the expression ofEGLN genes.

BACKGROUND OF THE INVENTION

Erythropoietin (EPO) is a hormone found in the plasma which regulatesred cell production by promoting erythroid differentiation andinitiating hemoglobin synthesis. The gene is in the EPO/TPO family andencodes a secreted, acidic glycosylated cytokine.

Recombinant human erythropoietin (EPO) has been used since 1986 to treatthe anemia of chronic and end-stage kidney disease (Eschbach, et al., N.Engl. J. Med. 1987 Jan. 8; 316(2):73-8). However, this treatment iscostly and requires parenteral administration. It has recently beenlinked to cardiovascular side effects (J. Bohlius et al., Lancet 373,1532 (2009) and antibodies which form against EPO can result in Pure RedCell Aplasia (PRCA), an uncommon condition which develops in associationwith a failure of the bone marrow to manufacture red blood cells,leaving patients with severe, treatment-resistant anemia (reported byCasadevall, et al, New England Journal of Medicine, Feb. 14, 2002).

In addition to its role as a kidney cytokine regulating hematopoiesis,EPO is also produced in the brain after oxidative or nitrosative stress.The transcription factor HIF1 (hypoxia inducible factor 1) is known toupregulate EPO following hypoxic stimuli (Digicaylioglu, M., Lipton, S.A. Nature 412: 641-647, 2001). This upregulation provides protectionagainst apoptosis of erythroid progenitors in bone marrow and alsoapoptosis of brain neurons (Siren, A.-L., et al., Proc. Nat. Acad. Sci.98: 4044-4049, 2001). Grimm et al. showed in the adult mouse retina thatacute hypoxia dose-dependently stimulates expression of EPO, fibroblastgrowth factor-2, and vascular endothelial growth factor via HIF1stabilization (Nature Med. 8: 718-724, 2002).

Further controlling the regulation of EPO production are a family ofprolyl hydroxylases, the PHD proteins, which act to regulate the HIFtranscription factors. PHD (prolyl hydroxylases) proteins belong to asuperfamily of several 2-oxoglutarate-dependent dioxygenases (KaelinJr., and Ratcliffe, Mol. Cell. 30, 393 (2008). In the mouse, these genesare known as EGLN1 (PHD2, prolyl hydroxylase domain-containing protein 2and by the synonyms hif-prolyl hydroxylase 2; hifph2; hph2; chromosome 1open reading frame 12; clorf12; sm20, rat, homolog of sm20; zinc fingermynd domain-containing protein 6; and zmynd6), EGLN2 (PHD1, prolylhydroxylase domain-containing protein 1; and by the synonyms hif-prolylhydroxylase 1; hifph1) and EGLN3 (PHD3 prolyl hydroxylasedomain-containing protein 3; and by the synonyms hif-prolyl hydroxylase3; hifph3). In an attempt to elucidate the function of PHD enzymes inhepatic EPO production, Minamishima et al., created knockout micelacking liver expression of PHD1, PHD2, PHD3, or combinations thereof(Mol. Cell. Biol. 29, 5729 (2009)).

Subsequent studies by Minamishima and Kaelin using the knock-out model,suggested that while hepatic inactivation of PHD 1, PHD2, or PHD3 alonedid not increase EPO or hematocrit values, loss of all three PHDsincreased both measurements (Science, 329, 407 and SupplementalInformation (2010)). According to Minamishima, questions remainregarding the promoters used and the role that PHD2 plays (and at whichdevelopmental stage) independent of the other two enzymes in theactivation of EPO production.

Double-stranded RNA molecules (dsRNA) have been shown to block geneexpression in a highly conserved regulatory mechanism known as RNAinterference (RNAi). This natural mechanism has now become the focus forthe development of a new class of pharmaceutical agents for treatingdisorders that are caused by the aberrant or unwanted regulation of agene.

Given the drawbacks of complete gene knockout and the inherent problemstranslating gene knockout to human therapy, the present inventioncontemplates the use of RNAi to effect gene modulation with improvedoutcomes in the production of erythropoietin.

During development the liver is the major source of EPO but over timeeventually the liver EPO is switched off and in normal healthy adultstheir kidney makes the EPO to support normal red blood cell production.However, two to four million Americans with renal disease suffer fromanemia due to impaired EPO production. If it is possible to turn onhepatic EPO using siRNA targeting EGLN genes the liver could now supplythe EPO required to support red blood cell production to compensate forthe damaged kidney function. Furthermore, using siRNA in LNPs it may bepossible to activate fetally expressed genes in liver by targetingnegative regulators of the pathway. This strategy could be used in thetreatment of many other diseases and not just exclusively anemia.

SUMMARY OF THE INVENTION

Described herein are compositions and methods that effect theRNA-induced silencing complex (RISC)-mediated cleavage of RNAtranscripts of one or more of the EGLN genes, such as in a cell ormammal Also described are compositions and methods for treatingpathological conditions and diseases caused by or associated with theexpression of said genes, such as anemia, hypoxia, neurologicalconditions including degeneration, renal disease or failure, and cancersincluding those of the blood, bone and marrow. It has been discoveredthat synergistic effects are seen upon the administration of a mix orplurality of iRNA agents collectively targeting all three EGLN genes.

As used herein, the term “iRNA” refers to one or more agents thatcontain RNA as that term is defined herein, and which mediates thetargeted cleavage of an RNA transcript via an RNA-induced silencingcomplex (RISC) pathway. In one embodiment, an iRNA as described hereineffects inhibition of expression of at least one EGLN gene in a cell ormammal. Alternatively, in another embodiment, an iRNA as describedherein activates EGLN expression in a cell or mammal. It should beunderstood that as used herein the term “EGLN” refers to any of the EGLNgenes in any mammalian species and having any of the synonyms referredto in the art. Where a specific species or gene variant is beingreferred to, the variant will be called out by name.

The iRNAs included in the compositions featured herein encompass a dsRNAhaving an RNA strand (the antisense strand) having a region that is 30nucleotides or less, generally 19-24 nucleotides in length, that issubstantially complementary to at least part of an mRNA transcript of anEGLN gene.

In one embodiment, an iRNA for inhibiting expression of an EGLN geneincludes at least two sequences that are complementary to each other.The iRNA includes a sense strand having a first sequence and anantisense strand having a second sequence. The antisense strand includesa nucleotide sequence that is substantially complementary to at leastpart of an mRNA encoding EGLN, and the region of complementarity is 30nucleotides or less, and at least 15 nucleotides in length. Generally,the iRNA is 19 to 24, e.g., 19 to 21 nucleotides in length. In someembodiments the iRNA is from about 15 to about 25 nucleotides in length,and in other embodiments the iRNA is from about 25 to about 30nucleotides in length. The iRNA, upon contacting with a cell expressingEGLN, inhibits the expression of an EGLN gene by at least 10%, at least20%, at least 25%, at least 30%, at least 35% or at least 40% or more,such as when assayed by a method as described herein. In one embodiment,where contacting is by a mix or plurality of EGLN iRNAs, the expressionof each EGLN gene is inhibited by at least 10%, at least 20%, at least25%, at least 30%, at least 35% or at least 40% or more and inhibitionneed not be the same for each EGLN targeted by the mix. For example, amix of iRNAs targeting EGLN1, 2 and 3 may result in inhibition ofexpression of EGLN1 by 10%, EGLN2 by 20% and EGLN3 by 10%. As such, themix inhibits EGLN expression by at least 10%. In one embodiment, theEGLN iRNA or iRNAs are formulated in a stable nucleic acid lipidparticle (SNALP).

The details of various embodiments of the invention are set forth in thedescription below. Other features, objects, and advantages of theinvention will be apparent from the description and the drawings, andfrom the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a histogram showing the in vitro screening results of the EGLN1, 2, and 3 genes. AD (duplex) numbers are those listed in Tables 2A-F.The additional digit listed in the figure after the decimal (“.”) pointis an internal tracking number and may be disregarded when makingreference to the duplexes listed in the tables.

FIG. 2 is a histogram showing the in vitro dose response screeningresults of the EGLN 1, 2, and 3 genes. AD (duplex) numbers are thoselisted in Tables 2A-F. The additional digit listed in the figure afterthe decimal (“.”) point is an internal tracking number and may bedisregarded when making reference to the duplexes listed in the tables.

FIG. 3 is a histogram showing the specificity of knockdown of EGLN genesby the iRNA agents of the invention. Panel 1 shows the specificity ofthe EGLN1 iRNA agent, AD-40894 for EGLN1 and the effect of the 3-iRNAmix. Panel 2 shows the specificity of the EGLN2 iRNA agent, AD-40773 forEGLN2 and the effect of the 3-iRNA mix. Panel 3 shows the specificity ofthe EGLN3 iRNA agent, AD-40758 for EGLN3 and the effect of the 3-iRNAmix.

FIG. 4 shows results from an ELISA assay. FIG. 4A shows a histogram ofEPO production in pg/mL Erythropoietin production upon treatment withEGLN dsRNA. FIG. 4B shows a histogram of the ELISA results of treatmentgroups PBS (1-4 and average), Luciferase control (AD1955) (1-5 andaverage) and the 3-iRNA mix of EGLN 1, 2 and 3 targeting agents,AD-40894, AD-40773 and AD40758, respectively (1-5 and average). Each bar(except for the averages) represents an individual animal.

FIG. 5 is a histogram showing the specificity of knockdown of EGLN genesby the iRNA agents of the invention in a dose response study (mg perkg). Panel 1 shows the specificity of the EGLN1 iRNA agent, AD-40894 forEGLN1. Panel 2 shows the specificity of the EGLN2 iRNA agent, AD-40773for EGLN2. Panel 3 shows the specificity of the EGLN3 iRNA agent,AD-40758 for EGLN3. Each panel also shows the knockdown of therespective EGLN gene using a dual iRNA agent mix (AD-04894 and AD-40758,“94/58” in amounts of 67% and 33% “0.67/0.33”)

FIG. 6 is a histogram of the Week 1 hematology results showingreticuloctye and RBC levels upon treatment with a composition comprisingan EGLN1-3 mix of iRNA agents.

FIG. 7 is a histogram of the Week 1 hematology results showinghemogolobin and hematocrit levels upon treatment with a compositioncomprising an EGLN1-3 mix of iRNA agents.

FIG. 8 is a histogram of the Week 2 hematology results showingreticuloctye and RBC levels upon treatment with a composition comprisingan EGLN1-3 mix of iRNA agents.

FIG. 9 is a histogram of the Week 2 hematology results showinghemogolobin and hematocrit levels upon treatment with a compositioncomprising an EGLN1-3 mix of iRNA agents.

FIG. 10 is a histogram showing the increase of EPO mRNA after 2 doses atday 10.

FIG. 11 is a histogram showing the specificity of knockdown of EGLNgenes by the iRNA agents of the invention in a dose response study (mgper kg). Panel 1 shows the specificity of the EGLN1 iRNA agent, AD-40894for EGLN1. Panel 2 shows the specificity of the EGLN2 iRNA agent,AD-40773 for EGLN2. Panel 3 shows the specificity of the EGLN3 iRNAagent, AD-40758 for EGLN3. Each panel also shows the knockdown of therespective EGLN gene using single iRNA agent mixes (AD-40894 is “EGLN1,”AD-40773 is “EGLN2” and AD-40758 is “EGLN3”), dual iRNA agent mixes(AD-40894 and AD-40773 is “EGLN 1+2,” AD-04894 and AD-40758 is “EGLN1+3,” AD-40773 and AD-40758 is “EGLN 2+3”) and a trip iRNA agent mix(AD-40894, AD-40773 and AD-40758 is “EGLN 1+2+3”).

FIG. 12 is a histogram showing the effects on erythropoietin productionby the iRNA agents of the invention in a dose response study (mg perkg). Panel 1 shows a histogram of the ELISA results of treatment groupsPBS, Luciferase control (AD1955), single iRNA agent mixes, dual iRNAagent mixes and triple iRNA mixture. Panel 2 shows the increase of EPOmRNA in the iRNA mixtures which contain the EGLN1 iRNA agent (AD-40773)from the treatment groups PBS, Luciferase control (AD1955), single iRNAagent mixes, dual iRNA agent mixes and triple iRNA mixture. It is to benoted that E1 means the same as EGLN1, E2 means the same as EGLN2 and E3means EGLN3.

FIG. 13 is a histogram of the hematology results showing hemogolobin,hematocrit, reticulocyte and red blood cell levels upon a two dosetreatment with a composition of single iRNA agents, dual iRNA agents ora triple iRNA agent mixture, a luciferase control iRNA agent and PBScontrol.

FIG. 14 is a histogram of the regulation of hepcidin upon a two dosetreatment with a composition of single iRNA agents, dual iRNA agents ora triple iRNA agent mixture, a luciferase control iRNA agent and PBScontrol.

FIG. 15 is a histogram showing tissue specificity in a dose responsestudy (mg per kg). Panel 1 shows a histogram of the results of treatmentgroups Luciferase control (AD1955 is “LUC”), and a triple iRNA mixture(AD-40894, AD-40773 and AD-40758 is “EGLN mix”) on EGLN1 found in theliver, kidney and spleen. Panel 2 shows a histogram of the results oftreatment groups Luciferase control (AD1955 is “LUC”), and a triple iRNAmixture (AD-40894, AD-40773 and AD-40758 is “EGLN mix”) on EGLN2 foundin the liver, kidney and spleen. Panel 3 shows a histogram of theresults of treatment groups Luciferase control (AD1955 is “LUC”), and atriple iRNA mixture (AD-40894, AD-40773 and AD-40758 is “EGLN mix”) onEGLN3 found in the liver, kidney and spleen. Panel 4 shows an increaseof EPO mRNA in the liver from the triple iRNA mixture (AD-40894,AD-40773 and AD-40758 is “EGLN mix”) as compared to the Luciferasecontrol (AD1955 is “LUC”) which was not seen in the kidney or spleen.The y-axis represents ratio of EPO to GAPDH mRNA levels in arbitraryunits.

FIG. 16 is a line graph showing the durable effects of a cocktail(AD-40894 at 0.375 mg/kg, AD-40773 at 0.75 mg/kg and AD-40758 at 0.375mg/kg) in a single dose injection or a double dose injection as comparedto a Luciferase control (AD1955). Panel 1 shows the levels of EPO foundafter a single or double injection as compared to the control (LUC).Panel 2 shows that the injection of the cocktail can increase the amounthematocrit in the mouse for about a month after a single injection.

FIG. 17 is a histogram showing knockdown of EGLN genes by the iRNAagents of the invention. Panel 1 shows the specificity of the EGLN1 iRNAagent, AD-40894 for EGLN1 (AD-40894), EGLN1-2 (mix of AD-40894 andAD-40773) and the effect of the 3-iRNA mix. Panel 2 shows thespecificity of the EGLN2 iRNA agent, AD-40773 for EGLN1 (AD-40894),EGLN1-2 (mix of AD-40894 and AD-40773) and the effect of the 3-iRNA mix.Panel 3 shows the specificity of the EGLN3 iRNA agent, AD-40758 forEGLN1 (AD-40894), EGLN1-2 (mix of AD-40894 and AD-40773) and the effectof the 3-iRNA mix.

FIG. 18 is a histogram a summary of the downregulation of hepcidin bythe iRNA agents of the invention.

FIG. 19 is a histogram showing the increase of EPO mRNA after 3 doses atday 12 in the animals who received the EGLN1-2-3 (mix of AD-40894,AD-40773 and AD-40758).

FIG. 20 is a scatter chart of the hematocrit levels for pre- andpost-dose of the iRNA agents of the invention. Panel 1 is the baselinehematocrit levels of the animals at day 0. Panel 2 is the hematocritlevels of the animals on day 12.

FIG. 21 is a histogram of the hematology results showing hemogolobin,hematocrit, reticulocyte and red blood cell levels upon a three dosetreatment with a composition of a single iRNA agent (EGLN1), dual iRNAagent (EGLN1+2) or a triple iRNA agent mixture (EGLN1+2+3), a luciferasecontrol iRNA agent, a PBS control and a SHAM control.

FIG. 22 is a scatter chart of the iron parameters of animals upon athree dose treatment with a composition of a single iRNA agent (EGLN1),dual iRNA agent (EGLN1-2) or a triple iRNA agent mixture (EGLN1-2-3), aluciferase control iRNA agent, a PBS control and a SHAM control. Panel 1shows the serum levels of iron in the animals. Panel 2 shows thetransferrin saturation (TSAT), which is the ratio of serum iron andtotal iron-binding capacity multiplied by 100, of the individualanimals. Panel 3 is the unsaturated iron binding capacity (UIBC) of theanimals. Panel 4 is the total iron binding capacity (TIBC) of theanimals. Panel 5 shows the ferritin level of the animals.

FIG. 23 shows the targeting of EglN genes rescues anemia caused by renalfailure. (A) Overview of 5/6 nephrectomy procedure and dosing schedule.(B and C) Hemoglobin (B) and Hematocrit (C) levels in mice treated asdepicted in (A).

FIG. 24 shows histograms of the hematologic data showing EPO and HAMP1mRNA values at day 12 in mice treated with the indicated siRNAs asdepicted in (A). HAMP1=hepcidin antimicrobrial peptide 1. mRNA levelswere normalized to actin mRNA and then to corresponding sham mRNA level.

FIG. 25 is a histogram showing the reduction of anemia in rats. Panel Ashows an effective knockdown of EGLN1 using the EGLN1/2 siRNAs of thepresent invention. Panel B shows an effective knockdown of EGLN2 usingthe EGLN1/2 siRNAs of the present invention. Panel C shows a decrease inhepcidin (HAMP1) levels in rats treated with the EGLN1/2 siRNAs of thepresent invention.

FIG. 26 shows bioluminescent images of HIF 1 alpha-Luc mice 72 hoursafter a single intravenous dose of LNPs targeting all three EglN familymembers or, as a negative control, green fluorescent protein (GFP).Total dose=1 mg/kg (0.33 mg/kg per family member).

DETAILED DESCRIPTION

Described herein are iRNAs and methods of using them for inhibiting theexpression of one or more EGLN genes in a cell or a mammal where theiRNA targets the one or more EGLN genes. Also described are compositionsand methods for treating pathological conditions and diseases caused byor associated with the expression of said genes, such as anemia,hypoxia, neurological conditions including degeneration, renal diseaseor failure, and cancers including those of the blood, bone and marrow.It has surprisingly been discovered that synergistic effects are seenupon the administration of a mix or plurality of iRNA agentscollectively targeting all three EGLN genes.

The iRNAs of the compositions featured herein include an RNA strand (theantisense strand) having a region which is 30 nucleotides or less inlength, i.e., 15-30 nucleotides in length, generally 19-24 nucleotidesin length, which region is substantially complementary to at least partof an mRNA transcript of an EGLN gene. The use of these iRNAs enablesthe targeted degradation of mRNAs of genes that are implicated inpathologies associated with EGLN expression in mammals and with thesignaling pathways involved in production of erythropoietin. Very lowdosages of EGLN iRNAs in particular can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of oneor more EGLN genes. Using cell-based assays, the present inventors havedemonstrated that iRNAs targeting EGLN can specifically and efficientlymediate RNAi, resulting in significant inhibition of expression of anEGLN gene. More surprising is the discovery by the present inventors ofa mix or cocktail of iRNA agents which can specifically target EGLN 1, 2and 3 and which can increase or stimulate erythropoietin production in acell or organism. Thus, methods and compositions including these iRNAsare useful for treating pathological processes that can be mediated bydown regulating EGLN genes or those which are associated with low EPOlevels. The following detailed description discloses how to make and usecompositions containing iRNAs to inhibit the expression of one or moreEGLN genes, as well as compositions and methods for treating diseasesand disorders caused by or modulated by the expression of this gene.Embodiments of the pharmaceutical compositions featured in the inventioninclude an iRNA having an antisense strand comprising a region which is30 nucleotides or less in length, generally 19-24 nucleotides in length,which region is substantially complementary to at least part of an RNAtranscript of an EGLN gene, together with a pharmaceutically acceptablecarrier. Embodiments of compositions featured in the invention alsoinclude an iRNA having an antisense strand having a region ofcomplementarity which is 30 nucleotides or less in length, generally19-24 nucleotides in length, and is substantially complementary to atleast part of an RNA transcript of an EGLN gene.

Accordingly, in some aspects, pharmaceutical compositions containing oneor more EGLN iRNA agents and a pharmaceutically acceptable carrier,methods of using the compositions to inhibit expression of an EGLN gene,and methods of using the pharmaceutical compositions to treat diseasescaused by expression of an EGLN gene are featured in the invention.

I. DEFINITIONS

For convenience, the meaning of certain terms and phrases used in thespecification, examples, and appended claims, are provided below. Ifthere is an apparent discrepancy between the usage of a term in otherparts of this specification and its definition provided in this section,the definition in this section shall prevail.

“G,” “C,” “A,” “T” and “U” each generally stand for a nucleotide thatcontains guanine, cytosine, adenine, thymidine and uracil as a base,respectively. However, it will be understood that the term“ribonucleotide” or “nucleotide” can also refer to a modifiednucleotide, as further detailed below, or a surrogate replacementmoiety. The skilled person is well aware that guanine, cytosine,adenine, and uracil may be replaced by other moieties withoutsubstantially altering the base pairing properties of an oligonucleotidecomprising a nucleotide bearing such replacement moiety. For example,without limitation, a nucleotide comprising inosine as its base may basepair with nucleotides containing adenine, cytosine, or uracil. Hence,nucleotides containing uracil, guanine, or adenine may be replaced inthe nucleotide sequences of dsRNA featured in the invention by anucleotide containing, for example, inosine. In another example, adenineand cytosine anywhere in the oligonucleotide can be replaced withguanine and uracil, respectively to form G-U Wobble base pairing withthe target mRNA. Sequences containing such replacement moieties aresuitable for the compositions and methods featured in the invention.

As used herein, “EGLN” (“EGL Nine Homolog”) refers to any one or all ofthe group of EGLN genes. In the mouse, these genes are known as EGLN1(PHD2, prolyl hydroxylase domain-containing protein 2 and by thesynonyms hif-prolyl hydroxylase 2; hifph2; hph2; chromosome 1 openreading frame 12; clorf12; sm20, rat, homolog of; sm20; zinc finger mynddomain-containing protein 6; and zmynd6), EGLN2 (PHD1, prolylhydroxylase domain-containing protein 1; and by the synonyms hif-prolylhydroxylase 1; hifph1) and EGLN3 (PHD3 prolyl hydroxylasedomain-containing protein 3; and by the synonyms hif-prolyl hydroxylase3; hifph3). The sequences of the mouse EGLN mRNA transcripts can befound at NM_(—)053207.2 (EGLN1; SEQ ID NO: 5), NM_(—)053208.4 (EGLN2;SEQ ID NO: 6) and NM_(—)028133.2 (EGLN3; SEQ ID NO: 7). The sequence ofa human EGLN mRNA transcripts can be found at NM_(—)022051.2 (EGLN1);NM_(—)053046.2 (EGLN2) and NM_(—)022073.3 (EGLN3).

As used herein, the term “iRNA” refers to an agent that contains RNA asthat term is defined herein, and which mediates the targeted cleavage ofan RNA transcript via an RNA-induced silencing complex (RISC) pathway.In one embodiment, an iRNA as described herein effects inhibition ofEGLN expression. Alternatively, in another embodiment, an iRNA asdescribed herein activates EGLN expression.

As used herein, the term “iRNA mix” or “iRNA cocktail” refers to acomposition that comprises more than one iRNA. The iRNA mixes orcocktails of the present invention may comprise one or more iRNA agentsto a single EGLN gene or may comprise one or more iRNA agents targetedto more than one EGLN gene. Where an iRNA mix or cocktail contains onlyiRNA agents targeting one or more EGLN genes, this mix may be referredto as an “EGLN mix” or “EGLN cocktail.”

As used herein, “target sequence” refers to a contiguous portion of thenucleotide sequence of an mRNA molecule formed during the transcriptionof an EGLN gene, including mRNA that is a product of RNA processing of aprimary transcription product. The target portion of the sequence willbe at least long enough to serve as a substrate for iRNA-directedcleavage at or near that portion. For example, the target sequence willgenerally be from 9-36 nucleotides in length, e.g., 15-30 nucleotides inlength, including all sub-ranges therebetween. As non-limiting examples,the target sequence can be from 15-30 nucleotides, 15-26 nucleotides,15-23 nucleotides, 15-22 nucleotides, 15-21 nucleotides, 15-20nucleotides, 15-19 nucleotides, 15-18 nucleotides, 15-17 nucleotides,18-30 nucleotides, 18-26 nucleotides, 18-23 nucleotides, 18-22nucleotides, 18-21 nucleotides, 18-20 nucleotides, 19-30 nucleotides,19-26 nucleotides, 19-23 nucleotides, 19-22 nucleotides, 19-21nucleotides, 19-20 nucleotides, 20-30 nucleotides, 20-26 nucleotides,20-25 nucleotides, 20-24 nucleotides, 20-23 nucleotides, 20-22nucleotides, 20-21 nucleotides, 21-30 nucleotides, 21-26 nucleotides,21-25 nucleotides, 21-24 nucleotides, 21-23 nucleotides, or 21-22nucleotides.

As used herein, the term “strand comprising a sequence” refers to anoligonucleotide comprising a chain of nucleotides that is described bythe sequence referred to using the standard nucleotide nomenclature.

As used herein, and unless otherwise indicated, the term“complementary,” when used to describe a first nucleotide sequence inrelation to a second nucleotide sequence, refers to the ability of anoligonucleotide or polynucleotide comprising the first nucleotidesequence to hybridize and form a duplex structure under certainconditions with an oligonucleotide or polynucleotide comprising thesecond nucleotide sequence, as will be understood by the skilled person.Such conditions can, for example, be stringent conditions, wherestringent conditions may include: 400 mM NaCl, 40 mM PIPES pH 6.4, 1 mMEDTA, 50° C. or 70° C. for 12-16 hours followed by washing. Otherconditions, such as physiologically relevant conditions as may beencountered inside an organism, can apply. The skilled person will beable to determine the set of conditions most appropriate for a test ofcomplementarity of two sequences in accordance with the ultimateapplication of the hybridized nucleotides.

Complementary sequences within an iRNA, e.g., within a dsRNA asdescribed herein, include base-pairing of the oligonucleotide orpolynucleotide comprising a first nucleotide sequence to anoligonucleotide or polynucleotide comprising a second nucleotidesequence over the entire length of one or both nucleotide sequences.Such sequences can be referred to as “fully complementary” with respectto each other herein. However, where a first sequence is referred to as“substantially complementary” with respect to a second sequence herein,the two sequences can be fully complementary, or they may form one ormore, but generally not more than 5, 4, 3 or 2 mismatched base pairsupon hybridization for a duplex up to 30 base pairs, while retaining theability to hybridize under the conditions most relevant to theirultimate application, e.g., inhibition of gene expression via a RISCpathway. However, where two oligonucleotides are designed to form, uponhybridization, one or more single stranded overhangs, such overhangsshall not be regarded as mismatches with regard to the determination ofcomplementarity. For example, a dsRNA comprising one oligonucleotide 21nucleotides in length and another oligonucleotide 23 nucleotides inlength, wherein the longer oligonucleotide comprises a sequence of 21nucleotides that is fully complementary to the shorter oligonucleotide,may yet be referred to as “fully complementary” for the purposesdescribed herein.

“Complementary” sequences, as used herein, may also include, or beformed entirely from, non-Watson-Crick base pairs and/or base pairsformed from non-natural and modified nucleotides, in as far as the aboverequirements with respect to their ability to hybridize are fulfilled.Such non-Watson-Crick base pairs includes, but are not limited to, G:UWobble or Hoogstein base pairing.

The terms “complementary,” “fully complementary” and “substantiallycomplementary” herein may be used with respect to the base matchingbetween the sense strand and the antisense strand of a dsRNA, or betweenthe antisense strand of an iRNA agent and a target sequence, as will beunderstood from the context of their use.

As used herein, a polynucleotide that is “substantially complementary toat least part of” a messenger RNA (mRNA) refers to a polynucleotide thatis substantially complementary to a contiguous portion of the mRNA ofinterest (e.g., an mRNA encoding an EGLN protein). For example, apolynucleotide is complementary to at least a part of an EGLN mRNA ifthe sequence is substantially complementary to a non-interrupted portionof an mRNA encoding EGLN.

The term “double-stranded RNA” or “dsRNA,” as used herein, refers to aniRNA that includes an RNA molecule or complex of molecules having ahybridized duplex region that comprises two anti-parallel andsubstantially complementary nucleic acid strands, which will be referredto as having “sense” and “antisense” orientations with respect to atarget RNA. The duplex region can be of any length that permits specificdegradation of a desired target RNA through a RISC pathway, but willtypically range from 9 to 36 base pairs in length, e.g., 15-30 basepairs in length. Considering a duplex between 9 and 36 base pairs, theduplex can be any length in this range, for example, 9, 10, 11, 12, 13,14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31,32, 33, 34, 35, or 36 and any sub-range therein between, including, butnot limited to 15-30 base pairs, 15-26 base pairs, 15-23 base pairs,15-22 base pairs, 15-21 base pairs, 15-20 base pairs, 15-19 base pairs,15-18 base pairs, 15-17 base pairs, 18-30 base pairs, 18-26 base pairs,18-23 base pairs, 18-22 base pairs, 18-21 base pairs, 18-20 base pairs,19-30 base pairs, 19-26 base pairs, 19-23 base pairs, 19-22 base pairs,19-21 base pairs, 19-20 base pairs, 20-30 base pairs, 20-26 base pairs,20-25 base pairs, 20-24 base pairs, 20-23 base pairs, 20-22 base pairs,20-21 base pairs, 21-30 base pairs, 21-26 base pairs, 21-25 base pairs,21-24 base pairs, 21-23 base pairs, or 21-22 base pairs. dsRNAsgenerated in the cell by processing with Dicer and similar enzymes aregenerally in the range of 19-22 base pairs in length. One strand of theduplex region of a dsDNA comprises a sequence that is substantiallycomplementary to a region of a target RNA. The two strands forming theduplex structure can be from a single RNA molecule having at least oneself-complementary region, or can be formed from two or more separateRNA molecules. Where the duplex region is formed from two strands of asingle molecule, the molecule can have a duplex region separated by asingle stranded chain of nucleotides (herein referred to as a “hairpinloop”) between the 3′-end of one strand and the 5′-end of the respectiveother strand forming the duplex structure. The hairpin loop can compriseat least one unpaired nucleotide; in some embodiments the hairpin loopcan comprise at least 3, at least 4, at least 5, at least 6, at least 7,at least 8, at least 9, at least 10, at least 20, at least 23 or moreunpaired nucleotides. Where the two substantially complementary strandsof a dsRNA are comprised by separate RNA molecules, those molecules neednot, but can be covalently connected. Where the two strands areconnected covalently by means other than a hairpin loop, the connectingstructure is referred to as a “linker.” The term “siRNA” is also usedherein to refer to a dsRNA as described above.

The skilled artisan will recognize that the term “RNA molecule” or“ribonucleic acid molecule” encompasses not only RNA molecules asexpressed or found in nature, but also analogs and derivatives of RNAcomprising one or more ribonucleotide/ribonucleoside analogs orderivatives as described herein or as known in the art. Strictlyspeaking, a “ribonucleoside” includes a nucleoside base and a ribosesugar, and a “ribonucleotide” is a ribonucleoside with one, two or threephosphate moieties. However, the terms “ribonucleoside” and“ribonucleotide” can be considered to be equivalent as used herein. TheRNA can be modified in the nucleobase structure or in theribose-phosphate backbone structure, e.g., as described herein below.However, the molecules comprising ribonucleoside analogs or derivativesmust retain the ability to form a duplex. As non-limiting examples, anRNA molecule can also include at least one modified ribonucleosideincluding but not limited to a 2′-O-methyl modified nucleostide, anucleoside comprising a 5′ phosphorothioate group, a terminal nucleosidelinked to a cholesteryl derivative or dodecanoic acid bisdecylamidegroup, a locked nucleoside, an abasic nucleoside, a 2′-deoxy-2′-fluoromodified nucleoside, a 2′-amino-modified nucleoside, 2′-alkyl-modifiednucleoside, morpholino nucleoside, a phosphoramidate or a non-naturalbase comprising nucleoside, or any combination thereof. Alternatively,an RNA molecule can comprise at least two modified ribonucleosides, atleast 3, at least 4, at least 5, at least 6, at least 7, at least 8, atleast 9, at least 10, at least 15, at least 20 or more, up to the entirelength of the dsRNA molecule. The modifications need not be the same foreach of such a plurality of modified ribonucleosides in an RNA molecule.In one embodiment, modified RNAs contemplated for use in methods andcompositions described herein are peptide nucleic acids (PNAs) that havethe ability to form the required duplex structure and that permit ormediate the specific degradation of a target RNA via a RISC pathway.

In one aspect, a modified ribonucleoside includes a deoxyribonucleoside.In such an instance, an iRNA agent can comprise one or moredeoxynucleosides, including, for example, a deoxynucleoside overhang(s),or one or more deoxynucleosides within the double stranded portion of adsRNA. However, it is self evident that under no circumstances is adouble stranded DNA molecule encompassed by the term “iRNA.”

In one aspect, an RNA interference agent includes a single stranded RNAthat interacts with a target RNA sequence to direct the cleavage of thetarget RNA. Without wishing to be bound by theory, long double strandedRNA introduced into plants and invertebrate cells is broken down intosiRNA by a Type III endonuclease known as Dicer (Sharp et al., GenesDev. 2001, 15:485). Dicer, a ribonuclease-III-like enzyme, processes thedsRNA into 19-23 base pair short interfering RNAs with characteristictwo base 3′ overhangs (Bernstein, et al., (2001) Nature 409:363). ThesiRNAs are then incorporated into an RNA-induced silencing complex(RISC) where one or more helicases unwind the siRNA duplex, enabling thecomplementary antisense strand to guide target recognition (Nykanen, etal., (2001) Cell 107:309). Upon binding to the appropriate target mRNA,one or more endonucleases within the RISC cleaves the target to inducesilencing (Elbashir, et al., (2001) Genes Dev. 15:188). Thus, in oneaspect the invention relates to a single stranded RNA that promotes theformation of a RISC complex to effect silencing of the target gene.

As used herein, the term “nucleotide overhang” refers to at least oneunpaired nucleotide that protrudes from the duplex structure of an iRNA,e.g., a dsRNA. For example, when a 3′-end of one strand of a dsRNAextends beyond the 5′-end of the other strand, or vice versa, there is anucleotide overhang. A dsRNA can comprise an overhang of at least onenucleotide; alternatively the overhang can comprise at least twonucleotides, at least three nucleotides, at least four nucleotides, atleast five nucleotides or more. A nucleotide overhang can comprise orconsist of a nucleotide/nucleoside analog, including adeoxynucleotide/nucleoside. The overhang(s) may be on the sense strand,the antisense strand or any combination thereof. Furthermore, thenucleotide(s) of an overhang can be present on the 5′ end, 3′ end orboth ends of either an antisense or sense strand of a dsRNA.

In one embodiment, the antisense strand of a dsRNA has a 1-10 nucleotideoverhang at the 3′ end and/or the 5′ end. In one embodiment, the sensestrand of a dsRNA has a 1-10 nucleotide overhang at the 3′ end and/orthe 5′ end. In another embodiment, one or more of the nucleotides in theoverhang is replaced with a nucleoside thiophosphate.

The terms “blunt” or “blunt ended” as used herein in reference to adsRNA mean that there are no unpaired nucleotides or nucleotide analogsat a given terminal end of a dsRNA, i.e., no nucleotide overhang. One orboth ends of a dsRNA can be blunt. Where both ends of a dsRNA are blunt,the dsRNA is said to be blunt ended. To be clear, a “blunt ended” dsRNAis a dsRNA that is blunt at both ends, i.e., no nucleotide overhang ateither end of the molecule. Most often such a molecule will bedouble-stranded over its entire length.

The term “antisense strand” or “guide strand” refers to the strand of aniRNA, e.g., a dsRNA, which includes a region that is substantiallycomplementary to a target sequence. As used herein, the term “region ofcomplementarity” refers to the region on the antisense strand that issubstantially complementary to a sequence, for example a targetsequence, as defined herein. Where the region of complementarity is notfully complementary to the target sequence, the mismatches may be in theinternal or terminal regions of the molecule. Generally, the mosttolerated mismatches are in the terminal regions, e.g., within 5, 4, 3,or 2 nucleotides of the 5′ and/or 3′ terminus.

The term “sense strand” or “passenger strand” as used herein, refers tothe strand of an iRNA that includes a region that is substantiallycomplementary to a region of the antisense strand as that term isdefined herein.

As used herein, the term “SNALP” refers to a stable nucleic acid-lipidparticle. A SNALP represents a vesicle of lipids coating a reducedaqueous interior comprising a nucleic acid such as an iRNA or a plasmidfrom which an iRNA is transcribed. SNALPs are described, e.g., in U.S.Patent Application Publication Nos. 20060240093, 20070135372, and inInternational Application No. WO 2009082817. These applications areincorporated herein by reference in their entirety.

“Introducing into a cell,” when referring to an iRNA, means facilitatingor effecting uptake or absorption into the cell, as is understood bythose skilled in the art. Absorption or uptake of an iRNA can occurthrough unaided diffusive or active cellular processes, or by auxiliaryagents or devices. The meaning of this term is not limited to cells invitro; an iRNA may also be “introduced into a cell,” wherein the cell ispart of a living organism. In such an instance, introduction into thecell will include the delivery to the organism. For example, for in vivodelivery, iRNA can be injected into a tissue site or administeredsystemically. In vivo delivery can also be by a beta-glucan deliverysystem, such as those described in U.S. Pat. Nos. 5,032,401 and5,607,677, and U.S. Publication No. 2005/0281781, which are herebyincorporated by reference in their entirety. In vitro introduction intoa cell includes methods known in the art such as electroporation andlipofection. Further approaches are described herein below or known inthe art.

As used herein, the term “modulate the expression of,” refers to at anleast partial “inhibition” or partial “activation” of one or more EGLNgene expression in a cell treated with an iRNA composition as describedherein compared to the expression of the one or more EGLN genes in anuntreated cell.

The terms “activate,” “enhance,” “up-regulate the expression of,”“increase the expression of,” and the like, in so far as they refer toan EGLN gene, herein refer to the at least partial activation of theexpression of an EGLN gene, as manifested by an increase in the amountof EGLN mRNA, which may be isolated from or detected in a first cell orgroup of cells in which an EGLN gene is transcribed and which has orhave been treated such that the expression of an EGLN gene is increased,as compared to a second cell or group of cells substantially identicalto the first cell or group of cells but which has or have not been sotreated (control cells).

In one embodiment, expression of an EGLN gene is activated by at leastabout 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or 50% by administrationof an iRNA as described herein. In some embodiments, an EGLN gene isactivated by at least about 60%, 70%, or 80% by administration of aniRNA featured in the invention. In some embodiments, expression of anEGLN gene is activated by at least about 85%, 90%, or 95% or more byadministration of an iRNA as described herein. In some embodiments, EGLNgene expression is increased by at least 1-fold, at least 2-fold, atleast 5-fold, at least 10-fold, at least 50-fold, at least 100-fold, atleast 500-fold, at least 1000 fold or more in cells treated with an iRNAas described herein compared to the expression in an untreated cell.Activation of expression by small dsRNAs is described, for example, inLi et al., 2006 Proc. Natl. Acad. Sci. U.S.A. 103:17337-42, and inUS20070111963 and US2005226848, each of which is incorporated herein byreference.

The terms “silence,” “inhibit the expression of,” “down-regulate theexpression of,” “suppress the expression of,” and the like, in so far asthey refer to an EGLN gene, herein refer to the at least partialsuppression of the expression of an EGLN gene, as manifested by areduction of the amount of EGLN mRNA which may be isolated from ordetected in a first cell or group of cells in which an EGLN gene istranscribed and which has or have been treated such that the expressionof an EGLN gene is inhibited, as compared to a second cell or group ofcells substantially identical to the first cell or group of cells butwhich has or have not been so treated (control cells). The degree ofinhibition is usually expressed in terms of

${\frac{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right) - \left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {treated}\mspace{14mu} {cells}} \right)}{\left( {{mRNA}\mspace{14mu} {in}\mspace{14mu} {control}\mspace{14mu} {cells}} \right)} \cdot 100}\%$

Alternatively, the degree of inhibition may be given in terms of areduction of a parameter that is functionally linked to EGLN geneexpression, e.g., the amount of protein encoded by an EGLN gene, or thenumber of cells displaying a certain phenotype, e.g., lack of ordecreased cytokine production. In principle, EGLN gene silencing may bedetermined in any cell expressing EGLN, either constitutively or bygenomic engineering, and by any appropriate assay. However, when areference is needed in order to determine whether a given iRNA inhibitsthe expression of an EGLN gene by a certain degree and therefore isencompassed by the instant invention, the assays provided in theExamples below shall serve as such reference.

For example, in certain instances, expression of an EGLN gene issuppressed by at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, or50% by administration of an iRNA featured in the invention. In someembodiments, an EGLN gene is suppressed by at least about 60%, 70%, or80% by administration of an iRNA featured in the invention. In someembodiments, an EGLN gene is suppressed by at least about 85%, 90%, 95%,98%, 99%, or more by administration of an iRNA as described herein.

As used herein in the context of EGLN expression, the terms “treat,”“treatment,” and the like, refer to relief from or alleviation ofpathological processes mediated by EGLN expression. In the context ofthe present invention insofar as it relates to any of the otherconditions recited herein below (other than pathological processesmediated by EGLN expression), the terms “treat,” “treatment,” and thelike mean to relieve or alleviate at least one symptom associated withsuch condition, or to slow or reverse the progression or anticipatedprogression of such condition, such as slowing the progression of amalignancy or cancer, treating anemia, hypoxia, neurological conditionsincluding degeneration, renal disease or failure, and cancers includingthose of the blood, bone and marrow.

By “lower” in the context of a disease marker or symptom is meant astatistically significant decrease in such level. The decrease can be,for example, at least 10%, at least 20%, at least 30%, at least 40% ormore, and is preferably down to a level accepted as within the range ofnormal for an individual without such disorder.

As used herein, the phrases “therapeutically effective amount” and“prophylactically effective amount” refer to an amount that provides atherapeutic benefit in the treatment, prevention, or management ofpathological processes mediated by EGLN expression or an overt symptomof pathological processes mediated by EGLN expression. In oneembodiment, a therapeutically effective amount is that amount of iRNAagent or agents which result in the increased production oferythropoietin in the system being treated. The specific amount that istherapeutically effective can be readily determined by an ordinarymedical practitioner, and may vary depending on factors known in theart, such as, for example, the type of pathological processes mediatedby EGLN expression, the patient's history and age, the stage ofpathological processes mediated by EGLN expression, and theadministration of other agents that inhibit pathological processesmediated by EGLN expression.

As used herein, a “pharmaceutical composition” comprises apharmacologically effective amount of an iRNA and a pharmaceuticallyacceptable carrier. As used herein, “pharmacologically effectiveamount,” “therapeutically effective amount” or simply “effective amount”refers to that amount of an iRNA effective to produce the intendedpharmacological, therapeutic or preventive result. For example, if agiven clinical treatment is considered effective when there is at leasta 10% reduction in a measurable parameter associated with a disease ordisorder, a therapeutically effective amount of a drug for the treatmentof that disease or disorder is the amount necessary to effect at least a10% reduction in that parameter. For example, a therapeuticallyeffective amount of an iRNA targeting EGLN can reduce EGLN proteinlevels by at least 10% or may result in the increase in EPO productionby at least 1%, 5%, 10% or more.

The term “pharmaceutically acceptable carrier” refers to a carrier foradministration of a therapeutic agent. Such carriers include, but arenot limited to, saline, buffered saline, dextrose, water, glycerol,ethanol, and combinations thereof. The term specifically excludes cellculture medium. For drugs administered orally, pharmaceuticallyacceptable carriers include, but are not limited to pharmaceuticallyacceptable excipients such as inert diluents, disintegrating agents,binding agents, lubricating agents, sweetening agents, flavoring agents,coloring agents and preservatives. Suitable inert diluents includesodium and calcium carbonate, sodium and calcium phosphate, and lactose,while corn starch and alginic acid are suitable disintegrating agents.Binding agents may include starch and gelatin, while the lubricatingagent, if present, will generally be magnesium stearate, stearic acid ortalc. If desired, the tablets may be coated with a material such asglyceryl monostearate or glyceryl distearate, to delay absorption in thegastrointestinal tract. Agents included in drug formulations aredescribed further herein below.

II. DOUBLE-STRANDED RIBONUCLEIC ACID (dsRNA)

Described herein are iRNA agents that inhibit the expression of one ormore EGLN genes. In one embodiment, the iRNA agent includesdouble-stranded ribonucleic acid (dsRNA) molecules for inhibiting theexpression of an EGLN gene in a cell or mammal, e.g., in a human havinganemia, hypoxia, neurological conditions including degeneration, renaldisease or failure, or cancers including those of the blood, bone andmarrow where the dsRNA includes an antisense strand having a region ofcomplementarity which is complementary to at least a part of an mRNAformed in the expression of an EGLN gene, and where the region ofcomplementarity is 30 nucleotides or less in length, generally 19-24nucleotides in length, and where the dsRNA, upon contact with a cellexpressing the EGLN gene, inhibits the expression of the EGLN gene by atleast 10% as assayed by, for example, a PCR or branched DNA (bDNA)-basedmethod, or by a protein-based method, such as by Western blot. In oneembodiment, the iRNA agent activates the expression of an EGLN gene in acell or mammal Expression of an EGLN gene in cell culture, such as inCOS cells, HeLa cells, primary hepatocytes, kidney cells, HEK-293 cells,MDCK cells, HepG2 cells, primary cultured cells or in a biologicalsample from a subject can be assayed by measuring EGLN mRNA levels, suchas by bDNA or TaqMan assay, or by measuring protein levels, such as byimmunofluorescence analysis, using, for example, Western Blotting orflowcytometric techniques.

A dsRNA includes two RNA strands that are sufficiently complementary tohybridize to form a duplex structure under conditions in which the dsRNAwill be used. One strand of a dsRNA (the antisense strand) includes aregion of complementarity that is substantially complementary, andgenerally fully complementary, to a target sequence, derived from thesequence of an mRNA formed during the expression of an EGLN gene. Theother strand (the sense strand) includes a region that is complementaryto the antisense strand, such that the two strands hybridize and form aduplex structure when combined under suitable conditions. Generally, theduplex structure is between 15 and 30 inclusive, more generally between18 and 25 inclusive, yet more generally between 19 and 24 inclusive, andmost generally between 19 and 21 base pairs in length, inclusive.Similarly, the region of complementarity to the target sequence isbetween 15 and 30 inclusive, more generally between 18 and 25 inclusive,yet more generally between 19 and 24 inclusive, and most generallybetween 19 and 21 nucleotides in length, inclusive. In some embodiments,the dsRNA is between 15 and 20 nucleotides in length, inclusive, and inother embodiments, the dsRNA is between 25 and 30 nucleotides in length,inclusive. As the ordinarily skilled person will recognize, the targetedregion of an RNA targeted for cleavage will most often be part of alarger RNA molecule, often an mRNA molecule. Where relevant, a “part” ofan mRNA target is a contiguous sequence of an mRNA target of sufficientlength to be a substrate for RNAi-directed cleavage (i.e., cleavagethrough a RISC pathway). dsRNAs having duplexes as short as 9 base pairscan, under some circumstances, mediate RNAi-directed RNA cleavage. Mostoften a target will be at least 15 nucleotides in length, preferably15-30 nucleotides in length.

One of skill in the art will also recognize that the duplex region is aprimary functional portion of a dsRNA, e.g., a duplex region of 9 to 36,e.g., 15-30 base pairs. Thus, in one embodiment, to the extent that itbecomes processed to a functional duplex of e.g., 15-30 base pairs thattargets a desired RNA for cleavage, an RNA molecule or complex of RNAmolecules having a duplex region greater than 30 base pairs is a dsRNA.Thus, an ordinarily skilled artisan will recognize that in oneembodiment, then, a miRNA is a dsRNA. In another embodiment, a dsRNA isnot a naturally occurring miRNA. In another embodiment, an iRNA agentuseful to target EGLN expression is not generated in the target cell bycleavage of a larger dsRNA.

A dsRNA as described herein may further include one or moresingle-stranded nucleotide overhangs. The dsRNA can be synthesized bystandard methods known in the art as further discussed below, e.g., byuse of an automated DNA synthesizer, such as are commercially availablefrom, for example, Biosearch, Applied Biosystems, Inc. In oneembodiment, an EGLN gene is a human EGLN gene. In another embodiment theEGLN gene is a mouse or a rat EGLN gene. In specific embodiments, thefirst sequence is a sense strand of a dsRNA that includes a sensesequence from Tables 2A-F and 6A-C, and the second sequence is selectedfrom the group consisting of the corresponding antisense sequences ofTables 2A-F and 6A-C. Alternative dsRNA agents that target elsewhere inthe target sequence provided in Tables 2A-F and 6A-C can readily bedetermined using the target sequence and the flanking EGLN sequence.

In one aspect, a dsRNA will include at least nucleotide sequences,whereby the sense strand is selected from the groups of sequencesprovided in Tables 2A-F and 6A-C, and the corresponding antisense strandof the sense strand selected from Tables 2A-F and 6A-C. In this aspect,one of the two sequences is complementary to the other of the twosequences, with one of the sequences being substantially complementaryto a sequence of an mRNA generated in the expression of an EGLN gene. Assuch, in this aspect, a dsRNA will include two oligonucleotides, whereone oligonucleotide is described as the sense strand in Tables 2A-F and6A-C, and the second oligonucleotide is described as the correspondingantisense strand of the sense strand from Tables 2A-F and 6A-C. Asdescribed elsewhere herein and as known in the art, the complementarysequences of a dsRNA can also be contained as self-complementary regionsof a single nucleic acid molecule, as opposed to being on separateoligonucleotides.

The skilled person is well aware that dsRNAs having a duplex structureof between 20 and 23, but specifically 21, base pairs have been hailedas particularly effective in inducing RNA interference (Elbashir et al.,EMBO 2001, 20:6877-6888). However, others have found that shorter orlonger RNA duplex structures can be effective as well. In theembodiments described above, by virtue of the nature of theoligonucleotide sequences provided in Tables 2A-F and 6A-C, dsRNAsdescribed herein can include at least one strand of a length ofminimally 21 nt. It can be reasonably expected that shorter duplexeshaving one of the sequences of Tables 2A-F and 6A-C minus only a fewnucleotides on one or both ends may be similarly effective as comparedto the dsRNAs described above. Hence, dsRNAs having a partial sequenceof at least 15, 16, 17, 18, 19, 20, or more contiguous nucleotides fromone of the sequences of Tables 2A-F and 6A-C, and differing in theirability to inhibit the expression of an EGLN gene by not more than 5,10, 15, 20, 25, or 30% inhibition from a dsRNA comprising the fullsequence, are contemplated according to the invention.

In addition, the RNAs provided in Tables 2A-F and 6A-C identify a sitein an EGLN transcript that is susceptible to RISC-mediated cleavage. Assuch, the present invention further features iRNAs that target withinone of such sequences. As used herein, an iRNA is said to target withina particular site of an RNA transcript if the iRNA promotes cleavage ofthe transcript anywhere within that particular site. Such an iRNA willgenerally include at least 15 contiguous nucleotides from one of thesequences provided in Tables 2A-F and 6A-C coupled to additionalnucleotide sequences taken from the region contiguous to the selectedsequence in an EGLN gene.

While a target sequence is generally 15-30 nucleotides in length, thereis wide variation in the suitability of particular sequences in thisrange for directing cleavage of any given target RNA. Various softwarepackages and the guidelines set out herein provide guidance for theidentification of optimal target sequences for any given gene target,but an empirical approach can also be taken in which a “window” or“mask” of a given size (as a non-limiting example, 21 nucleotides) isliterally or figuratively (including, e.g., in silico) placed on thetarget RNA sequence to identify sequences in the size range that mayserve as target sequences. By moving the sequence “window” progressivelyone nucleotide upstream or downstream of an initial target sequencelocation, the next potential target sequence can be identified, untilthe complete set of possible sequences is identified for any giventarget size selected. This process, coupled with systematic synthesisand testing of the identified sequences (using assays as describedherein or as known in the art) to identify those sequences that performoptimally can identify those RNA sequences that, when targeted with aniRNA agent, mediate the best inhibition of target gene expression. Thus,while the sequences identified, for example, in Tables 2A-F and 6A-Crepresent effective target sequences, it is contemplated that furtheroptimization of inhibition efficiency can be achieved by progressively“walking the window” one nucleotide upstream or downstream of the givensequences to identify sequences with equal or better inhibitioncharacteristics.

Further, it is contemplated that for any sequence identified, e.g., inTables 2A-F and 6A-C, further optimization could be achieved bysystematically either adding or removing nucleotides to generate longeror shorter sequences and testing those and sequences generated bywalking a window of the longer or shorter size up or down the target RNAfrom that point. Again, coupling this approach to generating newcandidate targets with testing for effectiveness of iRNAs based on thosetarget sequences in an inhibition assay as known in the art or asdescribed herein can lead to further improvements in the efficiency ofinhibition. Further still, such optimized sequences can be adjusted by,e.g., the introduction of modified nucleotides as described herein or asknown in the art, addition or changes in overhang, or othermodifications as known in the art and/or discussed herein to furtheroptimize the molecule (e.g., increasing serum stability or circulatinghalf-life, increasing thermal stability, enhancing transmembranedelivery, targeting to a particular location or cell type, increasinginteraction with silencing pathway enzymes, increasing release fromendosomes, etc.) as an expression inhibitor.

An iRNA as described herein can contain one or more mismatches to thetarget sequence. In one embodiment, an iRNA as described herein containsno more than 3 mismatches. If the antisense strand of the iRNA containsmismatches to a target sequence, it is preferable that the area ofmismatch not be located in the center of the region of complementarity.If the antisense strand of the iRNA contains mismatches to the targetsequence, it is preferable that the mismatch be restricted to be withinthe last 5 nucleotides from either the 5′ or 3′ end of the region ofcomplementarity. For example, for a 23 nucleotide iRNA agent RNA strandwhich is complementary to a region of an EGLN gene, the RNA strandgenerally does not contain any mismatch within the central 13nucleotides. The methods described herein or methods known in the artcan be used to determine whether an iRNA containing a mismatch to atarget sequence is effective in inhibiting the expression of an EGLNgene. Consideration of the efficacy of iRNAs with mismatches ininhibiting expression of an EGLN gene is important, especially if theparticular region of complementarity in an EGLN gene is known to havepolymorphic sequence variation within the population.

In one embodiment, at least one end of a dsRNA has a single-strandednucleotide overhang of 1 to 4, generally 1 or 2 nucleotides. dsRNAshaving at least one nucleotide overhang have unexpectedly superiorinhibitory properties relative to their blunt-ended counterparts. In yetanother embodiment, the RNA of an iRNA, e.g., a dsRNA, is chemicallymodified to enhance stability or other beneficial characteristics. Thenucleic acids featured in the invention may be synthesized and/ormodified by methods well established in the art, such as those describedin “Current protocols in nucleic acid chemistry,” Beaucage, S. L. et al.(Edrs.), John Wiley & Sons, Inc., New York, N.Y., USA, which is herebyincorporated herein by reference. Modifications include, for example,(a) end modifications, e.g., 5′ end modifications (phosphorylation,conjugation, inverted linkages, etc.) 3′ end modifications (conjugation,DNA nucleotides, inverted linkages, etc.), (b) base modifications, e.g.,replacement with stabilizing bases, destabilizing bases, or bases thatbase pair with an expanded repertoire of partners, removal of bases(abasic nucleotides), or conjugated bases, (c) sugar modifications(e.g., at the 2′ position or 4′ position) or replacement of the sugar,as well as (d) backbone modifications, including modification orreplacement of the phosphodiester linkages. Specific examples of RNAcompounds useful in this invention include, but are not limited to RNAscontaining modified backbones or no natural internucleoside linkages.RNAs having modified backbones include, among others, those that do nothave a phosphorus atom in the backbone. For the purposes of thisspecification, and as sometimes referenced in the art, modified RNAsthat do not have a phosphorus atom in their internucleoside backbone canalso be considered to be oligonucleosides. In particular embodiments,the modified RNA will have a phosphorus atom in its internucleosidebackbone.

Modified RNA backbones include, for example, phosphorothioates, chiralphosphorothioates, phosphorodithioates, phosphotriesters,aminoalkylphosphotriesters, methyl and other alkyl phosphonatesincluding 3′-alkylene phosphonates and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates having normal 3′-5′ linkages, 2′-5′ linked analogs ofthese, and those) having inverted polarity wherein the adjacent pairs ofnucleoside units are linked 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Varioussalts, mixed salts and free acid forms are also included.

Representative U.S. patents that teach the preparation of the abovephosphorus-containing linkages include, but are not limited to, U.S.Pat. Nos. 3,687,808; 4,469,863; 4,476,301; 5,023,243; 5,177,195;5,188,897; 5,264,423; 5,276,019; 5,278,302; 5,286,717; 5,321,131;5,399,676; 5,405,939; 5,453,496; 5,455,233; 5,466,677; 5,476,925;5,519,126; 5,536,821; 5,541,316; 5,550,111; 5,563,253; 5,571,799;5,587,361; 5,625,050; 6,028,188; 6,124,445; 6,160,109; 6,169,170;6,172,209; 6,239,265; 6,277,603; 6,326,199; 6,346,614; 6,444,423;6,531,590; 6,534,639; 6,608,035; 6,683,167; 6,858,715; 6,867,294;6,878,805; 7,015,315; 7,041,816; 7,273,933; 7,321,029; and U.S. Pat. No.RE39,464, each of which is herein incorporated by reference

Modified RNA backbones that do not include a phosphorus atom thereinhave backbones that are formed by short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatoms and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts.

Representative U.S. patents that teach the preparation of the aboveoligonucleosides include, but are not limited to, U.S. Pat. Nos.5,034,506; 5,166,315; 5,185,444; 5,214,134; 5,216,141; 5,235,033;5,64,562; 5,264,564; 5,405,938; 5,434,257; 5,466,677; 5,470,967;5,489,677; 5,541,307; 5,561,225; 5,596,086; 5,602,240; 5,608,046;5,610,289; 5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and,5,677,439, each of which is herein incorporated by reference.

In other RNA mimetics suitable or contemplated for use in iRNAs, boththe sugar and the internucleoside linkage, i.e., the backbone, of thenucleotide units are replaced with novel groups. The base units aremaintained for hybridization with an appropriate nucleic acid targetcompound. One such oligomeric compound, an RNA mimetic that has beenshown to have excellent hybridization properties, is referred to as apeptide nucleic acid (PNA). In PNA compounds, the sugar backbone of anRNA is replaced with an amide containing backbone, in particular anaminoethylglycine backbone. The nucleobases are retained and are bounddirectly or indirectly to aza nitrogen atoms of the amide portion of thebackbone. Representative U.S. patents that teach the preparation of PNAcompounds include, but are not limited to, U.S. Pat. Nos. 5,539,082;5,714,331; and 5,719,262, each of which is herein incorporated byreference. Further teaching of PNA compounds can be found, for example,in Nielsen et al., Science, 1991, 254, 1497-1500.

Some embodiments featured in the invention include RNAs withphosphorothioate backbones and oligonucleosides with heteroatombackbones, and in particular —CH₂—NH—CH₂—, —CH₂—N(CH₃)—O—CH₂— [known asa methylene (methylimino) or MMI backbone], —CH₂—O—N(CH₃)—CH₂—,—CH₂—N(CH₃)—N(CH₃)—CH₂— and —N(CH₃)—CH₂—CH₂—[wherein the nativephosphodiester backbone is represented as —O—P—O—CH₂—] of theabove-referenced U.S. Pat. No. 5,489,677, and the amide backbones of theabove-referenced U.S. Pat. No. 5,602,240. In some embodiments, the RNAsfeatured herein have morpholino backbone structures of theabove-referenced U.S. Pat. No. 5,034,506.

Modified RNAs may also contain one or more substituted sugar moieties.The iRNAs, e.g., dsRNAs, featured herein can include one of thefollowing at the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, orN-alkenyl; O-, S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl,alkenyl and alkynyl may be substituted or unsubstituted C₁ to C₁₀ alkylor C₂ to C₁₀ alkenyl and alkynyl. Exemplary suitable modificationsinclude O[(CH₂)_(m)O]_(m)CH₃, O(CH₂)._(n)OCH₃, O(CH₂)_(n)NH₂,O(CH₂)_(n)CH₃, O(CH₂)_(n)ONH₂, and O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂, where nand m are from 1 to about 10. In other embodiments, dsRNAs include oneof the following at the 2′ position: C₁ to C₁₀ lower alkyl, substitutedlower alkyl, alkaryl, aralkyl, O-alkaryl or O-aralkyl, SH, SCH₃, OCN,Cl, Br, CN, CF₃, OCF₃, SOCH₃, SO₂CH₃, ONO₂, NO₂, N₃, NH₂,heterocycloalkyl, heterocycloalkaryl, aminoalkylamino, polyalkylamino,substituted silyl, an RNA cleaving group, a reporter group, anintercalator, a group for improving the pharmacokinetic properties of aniRNA, or a group for improving the pharmacodynamic properties of aniRNA, and other substituents having similar properties. In someembodiments, the modification includes a 2′-methoxyethoxy(2′-O—CH₂CH₂OCH₃, also known as 2′-O-(2-methoxyethyl) or 2′-MOE) (Martinet al., Hely. Chim. Acta, 1995, 78:486-504) i.e., an alkoxy-alkoxygroup. Another exemplary modification is 2′-dimethylaminooxyethoxy,i.e., a O(CH₂)₂ON(CH₃)₂ group, also known as 2′-DMAOE, as described inexamples herein below, and 2′-dimethylaminoethoxyethoxy (also known inthe art as 2′-β-dimethylaminoethoxyethyl or 2′-DMAEOE), i.e.,2′-O—CH₂—O—CH₂—N(CH₂)₂, also described in examples herein below.

Other modifications include 2′-methoxy (2′-OCH₃), 2′-aminopropoxy(2′-OCH₂CH₂CH₂NH₂) and 2′-fluoro (2′-F). Similar modifications may alsobe made at other positions on the RNA of an iRNA, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkeddsRNAs and the 5′ position of 5′ terminal nucleotide. iRNAs may alsohave sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. Representative U.S. patents that teach thepreparation of such modified sugar structures include, but are notlimited to, U.S. Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044;5,393,878; 5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811;5,576,427; 5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873;5,646,265; 5,658,873; 5,670,633; and 5,700,920, certain of which arecommonly owned with the instant application, and each of which is hereinincorporated by reference.

An iRNA may also include nucleobase (often referred to in the art simplyas “base”) modifications or substitutions. As used herein, “unmodified”or “natural” nucleobases include the purine bases adenine (A) andguanine (G), and the pyrimidine bases thymine (T), cytosine (C) anduracil (U). Modified nucleobases include other synthetic and naturalnucleobases such as 5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine,xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkylderivatives of adenine and guanine, 2-propyl and other alkyl derivativesof adenine and guanine, 2-thiouracil, 2-thiothymine and 2-thiocytosine,5-halouracil and cytosine, 5-propynyl uracil and cytosine, 6-azo uracil,cytosine and thymine, 5-uracil (pseudouracil), 4-thiouracil, 8-halo,8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl anal other 8-substitutedadenines and guanines, 5-halo, particularly 5-bromo, 5-trifluoromethyland other 5-substituted uracils and cytosines, 7-methylguanine and7-methyladenine, 8-azaguanine and 8-azaadenine, 7-deazaguanine and7-daazaadenine and 3-deazaguanine and 3-deazaadenine. Furthernucleobases include those disclosed in U.S. Pat. No. 3,687,808, thosedisclosed in Modified Nucleosides in Biochemistry, Biotechnology andMedicine, Herdewijn, P. ed. Wiley-VCH, 2008; those disclosed in TheConcise Encyclopedia Of Polymer Science And Engineering, pages 858-859,Kroschwitz, J. L, ed. John Wiley & Sons, 1990, these disclosed byEnglisch et al., Angewandte Chemie, International Edition, 1991, 30,613, and those disclosed by Sanghvi, Y S., Chapter 15, dsRNA Researchand Applications, pages 289-302, Crooke, S. T. and Lebleu, B., Ed., CRCPress, 1993. Certain of these nucleobases are particularly useful forincreasing the binding affinity of the oligomeric compounds featured inthe invention. These include 5-substituted pyrimidines, 6-azapyrimidinesand N-2, N-6 and 0-6 substituted purines, including2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine substitutions have been shown to increase nucleic acidduplex stability by 0.6-1.2° C. (Sanghvi, Y. S., Crooke, S. T. andLebleu, B., Eds., dsRNA Research and Applications, CRC Press, BocaRaton, 1993, pp. 276-278) and are exemplary base substitutions, evenmore particularly when combined with 2′-O-methoxyethyl sugarmodifications.

Representative U.S. patents that teach the preparation of certain of theabove noted modified nucleobases as well as other modified nucleobasesinclude, but are not limited to, the above noted U.S. Pat. No.3,687,808, as well as U.S. Pat. Nos. 4,845,205; 513,030; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121; 5,596,091;5,614,617; 5,681,941; 6,015,886; 6,147,200; 6,166,197; 6,222,025;6,235,887; 6,380,368; 6,528,640; 6,639,062; 6,617,438; 7,045,610;7,427,672; and 7,495,088, each of which is herein incorporated byreference, and U.S. Pat. No. 5,750,692, also herein incorporated byreference.

The RNA of an iRNA can also be modified to include one or more lockednucleic acids (LNA). A locked nucleic acid is a nucleotide having amodified ribose moiety in which the ribose moiety comprises an extrabridge connecting the 2′ and 4′ carbons. This structure effectively“locks” the ribose in the 3′-endo structural conformation. The additionof locked nucleic acids to siRNAs has been shown to increase siRNAstability in serum, and to reduce off-target effects (Elmen, J. et al.,(2005) Nucleic Acids Research 33(1):439-447; Mook, O R. et al., (2007)Mol Canc Ther 6(3):833-843; Grunweller, A. et al., (2003) Nucleic AcidsResearch 31(12):3185-3193).

Representative U.S. patents that teach the preparation of locked nucleicacid nucleotides include, but are not limited to, the following: U.S.Pat. Nos. 6,268,490; 6,670,461; 6,794,499; 6,998,484; 7,053,207;7,084,125; and 7,399,845, each of which is herein incorporated byreference in its entirety.

Another modification of the RNA of an iRNA featured in the inventioninvolves chemically linking to the RNA one or more ligands, moieties orconjugates that enhance the activity, cellular distribution or cellularuptake of the iRNA. Such moieties include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acid. Sci. USA, 1989, 86: 6553-6556), cholic acid (Manoharan et al.,Biorg. Med. Chem. Let., 1994, 4:1053-1060), a thioether, e.g.,beryl-5-tritylthiol (Manoharan et al., Ann. N.Y. Acad. Sci., 1992,660:306-309; Manoharan et al., Biorg. Med. Chem. Let., 1993,3:2765-2770), a thiocholesterol (Oberhauser et al., Nucl. Acids Res.,1992, 20:533-538), an aliphatic chain, e.g., dodecandiol or undecylresidues (Saison-Behmoaras et al., EMBO J, 1991, 10:1111-1118; Kabanovet al., FEBS Lett., 1990, 259:327-330; Svinarchuk et al., Biochimie,1993, 75:49-54), a phospholipid, e.g., di-hexadecyl-rac-glycerol ortriethyl-ammonium 1,2-di-O-hexadecyl-rac-glycero-3-phosphonate(Manoharan et al., Tetrahedron Lett., 1995, 36:3651-3654; Shea et al.,Nucl. Acids Res., 1990, 18:3777-3783), a polyamine or a polyethyleneglycol chain (Manoharan et al., Nucleosides & Nucleotides, 1995,14:969-973), or adamantane acetic acid (Manoharan et al., TetrahedronLett., 1995, 36:3651-3654), a palmityl moiety (Mishra et al., Biochim.Biophys. Acta, 1995, 1264:229-237), or an octadecylamine orhexylamino-carbonyloxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277:923-937).

In one embodiment, a ligand alters the distribution, targeting orlifetime of an iRNA agent into which it is incorporated. In preferredembodiments a ligand provides an enhanced affinity for a selectedtarget, e.g., molecule, cell or cell type, compartment, e.g., a cellularor organ compartment, tissue, organ or region of the body, as, e.g.,compared to a species absent such a ligand. Preferred ligands will nottake part in duplex pairing in a duplexed nucleic acid.

Ligands can include a naturally occurring substance, such as a protein(e.g., human serum albumin (HSA), low-density lipoprotein (LDL), orglobulin); carbohydrate (e.g., a dextran, pullulan, chitin, chitosan,inulin, cyclodextrin or hyaluronic acid); or a lipid. The ligand mayalso be a recombinant or synthetic molecule, such as a syntheticpolymer, e.g., a synthetic polyamino acid. Examples of polyamino acidsinclude polyamino acid is a polylysine (PLL), poly L-aspartic acid, polyL-glutamic acid, styrene-maleic acid anhydride copolymer,poly(L-lactide-co-glycolied) copolymer, divinyl ether-maleic anhydridecopolymer, N-(2-hydroxypropyl)methacrylamide copolymer (HMPA),polyethylene glycol (PEG), polyvinyl alcohol (PVA), polyurethane,poly(2-ethylacryllic acid), N-isopropylacrylamide polymers, orpolyphosphazine. Example of polyamines include: polyethylenimine,polylysine (PLL), spermine, spermidine, polyamine,pseudopeptide-polyamine, peptidomimetic polyamine, dendrimer polyamine,arginine, amidine, protamine, cationic lipid, cationic porphyrin,quaternary salt of a polyamine, or an alpha helical peptide.

Ligands can also include targeting groups, e.g., a cell or tissuetargeting agent, e.g., a lectin, glycoprotein, lipid or protein, e.g.,an antibody, that binds to a specified cell type such as a kidney cell.A targeting group can be a thyrotropin, melanotropin, lectin,glycoprotein, surfactant protein A, Mucin carbohydrate, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, multivalent fucose,glycosylated polyaminoacids, multivalent galactose, transferrin,bisphosphonate, polyglutamate, polyaspartate, a lipid, cholesterol, asteroid, bile acid, folate, vitamin B12, biotin, or an RGD peptide orRGD peptide mimetic.

Other examples of ligands include dyes, intercalating agents (e.g.acridines), cross-linkers (e.g. psoralene, mitomycin C), porphyrins(TPPC4, texaphyrin, Sapphyrin), polycyclic aromatic hydrocarbons (e.g.,phenazine, dihydrophenazine), artificial endonucleases (e.g. EDTA),lipophilic molecules, e.g., cholesterol, cholic acid, adamantane aceticacid, 1-pyrene butyric acid, dihydrotestosterone,1,3-Bis-O(hexadecyl)glycerol, geranyloxyhexyl group, hexadecylglycerol,borneol, menthol, 1,3-propanediol, heptadecyl group, palmitic acid,myristic acid,O3-(oleoyl)lithocholic acid, O3-(oleoyl)cholenic acid,dimethoxytrityl, or phenoxazine) and peptide conjugates (e.g.,antennapedia peptide, Tat peptide), alkylating agents, phosphate, amino,mercapto, PEG (e.g., PEG-40K), MPEG, [MPEG]₂, polyamino, alkyl,substituted alkyl, radiolabeled markers, enzymes, haptens (e.g. biotin),transport/absorption facilitators (e.g., aspirin, vitamin E, folicacid), synthetic ribonucleases (e.g., imidazole, bisimidazole,histamine, imidazole clusters, acridine-imidazole conjugates, Eu3+complexes of tetraazamacrocycles), dinitrophenyl, HRP, or AP.

Ligands can be proteins, e.g., glycoproteins, or peptides, e.g.,molecules having a specific affinity for a co-ligand, or antibodiese.g., an antibody, that binds to a specified cell type such as a cancercell, endothelial cell, or bone cell. Ligands may also include hormonesand hormone receptors. They can also include non-peptidic species, suchas lipids, lectins, carbohydrates, vitamins, cofactors, multivalentlactose, multivalent galactose, N-acetyl-galactosamine,N-acetyl-gulucosamine multivalent mannose, or multivalent fucose. Theligand can be, for example, a lipopolysaccharide, an activator of p38MAP kinase, or an activator of NF-κB.

The ligand can be a substance, e.g., a drug, which can increase theuptake of the iRNA agent into the cell, for example, by disrupting thecell's cytoskeleton, e.g., by disrupting the cell's microtubules,microfilaments, and/or intermediate filaments. The drug can be, forexample, taxon, vincristine, vinblastine, cytochalasin, nocodazole,japlakinolide, latrunculin A, phalloidin, swinholide A, indanocine, ormyoservin.

In one ligand, the ligand is a lipid or lipid-based molecule. Such alipid or lipid-based molecule preferably binds a serum protein, e.g.,human serum albumin (HSA). An HSA binding ligand allows for distributionof the conjugate to a target tissue, e.g., a non-kidney target tissue ofthe body. For example, the target tissue can be the liver, includingparenchymal cells of the liver. Other molecules that can bind HSA canalso be used as ligands. For example, neproxin or aspirin can be used. Alipid or lipid-based ligand can (a) increase resistance to degradationof the conjugate, (b) increase targeting or transport into a target cellor cell membrane, and/or (c) can be used to adjust binding to a serumprotein, e.g., HSA.

A lipid based ligand can be used to modulate, e.g., control the bindingof the conjugate to a target tissue. For example, a lipid or lipid-basedligand that binds to HSA more strongly will be less likely to betargeted to the kidney and therefore less likely to be cleared from thebody. A lipid or lipid-based ligand that binds to HSA less strongly canbe used to target the conjugate to the kidney.

In a preferred embodiment, the lipid based ligand binds HSA. Preferably,it binds HSA with a sufficient affinity such that the conjugate will bepreferably distributed to a non-kidney tissue. However, it is preferredthat the affinity not be so strong that the HSA-ligand binding cannot bereversed.

In another preferred embodiment, the lipid based ligand binds HSA weaklyor not at all, such that the conjugate will be preferably distributed tothe kidney. Other moieties that target to kidney cells can also be usedin place of or in addition to the lipid based ligand.

In another aspect, the ligand is a moiety, e.g., a vitamin, which istaken up by a target cell, e.g., a proliferating cell. These areparticularly useful for treating disorders characterized by unwantedcell proliferation, e.g., of the malignant or non-malignant type, e.g.,cancer cells. Exemplary vitamins include vitamin A, E, and K. Otherexemplary vitamins include are B vitamin, e.g., folic acid, B12,riboflavin, biotin, pyridoxal or other vitamins or nutrients taken up bycancer cells. Also included are HSA and low density lipoprotein (LDL).

In another aspect, the ligand is a cell-permeation agent, preferably ahelical cell-permeation agent. Preferably, the agent is amphipathic. Anexemplary agent is a peptide such as tat or antennopedia. If the agentis a peptide, it can be modified, including a peptidylmimetic,invertomers, non-peptide or pseudo-peptide linkages, and use of D-aminoacids. The helical agent is preferably an alpha-helical agent, whichpreferably has a lipophilic and a lipophobic phase.

The ligand can be a peptide or peptidomimetic. A peptidomimetic (alsoreferred to herein as an oligopeptidomimetic) is a molecule capable offolding into a defined three-dimensional structure similar to a naturalpeptide. The attachment of peptide and peptidomimetics to iRNA agentscan affect pharmacokinetic distribution of the iRNA, such as byenhancing cellular recognition and absorption. The peptide orpeptidomimetic moiety can be about 5-50 amino acids long, e.g., about 5,10, 15, 20, 25, 30, 35, 40, 45, or 50 amino acids long.

A peptide or peptidomimetic can be, for example, a cell permeationpeptide, cationic peptide, amphipathic peptide, or hydrophobic peptide(e.g., consisting primarily of Tyr, Trp or Phe). The peptide moiety canbe a dendrimer peptide, constrained peptide or crosslinked peptide. Inanother alternative, the peptide moiety can include a hydrophobicmembrane translocation sequence (MTS). An exemplary hydrophobicMTS-containing peptide is RFGF having the amino acid sequenceAAVALLPAVLLALLAP (SEQ ID NO:1). An RFGF analogue (e.g., amino acidsequence AALLPVLLAAP (SEQ ID NO:2)) containing a hydrophobic MTS canalso be a targeting moiety. The peptide moiety can be a “delivery”peptide, which can carry large polar molecules including peptides,oligonucleotides, and protein across cell membranes. For example,sequences from the HIV Tat protein (GRKKRRQRRRPPQ (SEQ ID NO:3)) and theDrosophila antennapedia protein (RQIKIWFQNRRMKWKK (SEQ ID NO: 4)) havebeen found to be capable of functioning as delivery peptides. A peptideor peptidomimetic can be encoded by a random sequence of DNA, such as apeptide identified from a phage-display library, orone-bead-one-compound (OBOC) combinatorial library (Lam et al., Nature,354:82-84, 1991). Preferably the peptide or peptidomimetic tethered to adsRNA agent via an incorporated monomer unit is a cell targeting peptidesuch as an arginine-glycine-aspartic acid (RGD)-peptide, or RGD mimic Apeptide moiety can range in length from about 5 amino acids to about 40amino acids. The peptide moieties can have a structural modification,such as to increase stability or direct conformational properties. Anyof the structural modifications described below can be utilized.

An RGD peptide moiety can be used to target a tumor cell, such as anendothelial tumor cell or a breast cancer tumor cell (Zitzmann et al.,Cancer Res., 62:5139-43, 2002). An RGD peptide can facilitate targetingof an dsRNA agent to tumors of a variety of other tissues, including thelung, kidney, spleen, or liver (Aoki et al., Cancer Gene Therapy8:783-787, 2001). Preferably, the RGD peptide will facilitate targetingof an iRNA agent to the kidney. The RGD peptide can be linear or cyclic,and can be modified, e.g., glycosylated or methylated to facilitatetargeting to specific tissues. For example, a glycosylated RGD peptidecan deliver a iRNA agent to a tumor cell expressing α_(v)β₃ (Haubner etal., Jour. Nucl. Med., 42:326-336, 2001).

A “cell permeation peptide” is capable of permeating a cell, e.g., amicrobial cell, such as a bacterial or fungal cell, or a mammalian cell,such as a human cell. A microbial cell-permeating peptide can be, forexample, an α-helical linear peptide (e.g., LL-37 or Ceropin P1), adisulfide bond-containing peptide (e.g., α-defensin, (β-defensin orbactenecin), or a peptide containing only one or two dominating aminoacids (e.g., PR-39 or indolicidin). A cell permeation peptide can alsoinclude a nuclear localization signal (NLS). For example, a cellpermeation peptide can be a bipartite amphipathic peptide, such as MPG,which is derived from the fusion peptide domain of HIV-1 gp41 and theNLS of SV40 large T antigen (Simeoni et al., Nucl. Acids Res.31:2717-2724, 2003).

Representative U.S. patents that teach the preparation of RNA conjugatesinclude, but are not limited to, U.S. Pat. Nos. 4,828,979; 4,948,882;5,218,105; 5,525,465; 5,541,313; 5,545,730; 5,552,538; 5,578,717,5,580,731; 5,591,584; 5,109,124; 5,118,802; 5,138,045; 5,414,077;5,486,603; 5,512,439; 5,578,718; 5,608,046; 4,587,044; 4,605,735;4,667,025; 4,762,779; 4,789,737; 4,824,941; 4,835,263; 4,876,335;4,904,582; 4,958,013; 5,082,830; 5,112,963; 5,214,136; 5,082,830;5,112,963; 5,214,136; 5,245,022; 5,254,469; 5,258,506; 5,262,536;5,272,250; 5,292,873; 5,317,098; 5,371,241, 5,391,723; 5,416,203,5,451,463; 5,510,475; 5,512,667; 5,514,785; 5,565,552; 5,567,810;5,574,142; 5,585,481; 5,587,371; 5,595,726; 5,597,696; 5,599,923;5,599,928 and 5,688,941; 6,294,664; 6,320,017; 6,576,752; 6,783,931;6,900,297; 7,037,646; each of which is herein incorporated by reference.

It is not necessary for all positions in a given compound to beuniformly modified, and in fact more than one of the aforementionedmodifications may be incorporated in a single compound or even at asingle nucleoside within an iRNA. The present invention also includesiRNA compounds that are chimeric compounds. “Chimeric” iRNA compounds or“chimeras,” in the context of this invention, are iRNA compounds,preferably dsRNAs, which contain two or more chemically distinctregions, each made up of at least one monomer unit, i.e., a nucleotidein the case of a dsRNA compound. These iRNAs typically contain at leastone region wherein the RNA is modified so as to confer upon the iRNAincreased resistance to nuclease degradation, increased cellular uptake,and/or increased binding affinity for the target nucleic acid. Anadditional region of the iRNA may serve as a substrate for enzymescapable of cleaving RNA:DNA or RNA:RNA hybrids. By way of example, RNaseH is a cellular endonuclease which cleaves the RNA strand of an RNA:DNAduplex. Activation of RNase H, therefore, results in cleavage of the RNAtarget, thereby greatly enhancing the efficiency of iRNA inhibition ofgene expression. Consequently, comparable results can often be obtainedwith shorter iRNAs when chimeric dsRNAs are used, compared tophosphorothioate deoxy dsRNAs hybridizing to the same target region.Cleavage of the RNA target can be routinely detected by gelelectrophoresis and, if necessary, associated nucleic acid hybridizationtechniques known in the art.

In certain instances, the RNA of an iRNA can be modified by a non-ligandgroup. A number of non-ligand molecules have been conjugated to iRNAs inorder to enhance the activity, cellular distribution or cellular uptakeof the iRNA, and procedures for performing such conjugations areavailable in the scientific literature. Such non-ligand moieties haveincluded lipid moieties, such as cholesterol (Kubo, T. et al., Biochem.Biophys. Res. Comm., 2007, 365(1):54-61; Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989, 86:6553), cholic acid (Manoharan et al., Bioorg.Med. Chem. Lett., 1994, 4:1053), a thioether, e.g., hexyl-5-tritylthiol(Manoharan et al., Ann. N.Y. Acad. Sci., 1992, 660:306; Manoharan etal., Bioorg. Med. Chem. Let., 1993, 3:2765), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20:533), an aliphatic chain,e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al., EMBO J.,1991, 10:111; Kabanov et al., FEBS Lett., 1990, 259:327; Svinarchuk etal., Biochimie, 1993, 75:49), a phospholipid, e.g.,di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36:3651; Shea et al., Nucl. Acids Res., 1990,18:3777), a polyamine or a polyethylene glycol chain (Manoharan et al.,Nucleosides & Nucleotides, 1995, 14:969), or adamantane acetic acid(Manoharan et al., Tetrahedron Lett., 1995, 36:3651), a palmityl moiety(Mishra et al., Biochim. Biophys. Acta, 1995, 1264:229), or anoctadecylamine or hexylamino-carbonyl-oxycholesterol moiety (Crooke etal., J. Pharmacol. Exp. Ther., 1996, 277:923). Representative UnitedStates patents that teach the preparation of such RNA conjugates havebeen listed above. Typical conjugation protocols involve the synthesisof an RNAs bearing an aminolinker at one or more positions of thesequence. The amino group is then reacted with the molecule beingconjugated using appropriate coupling or activating reagents. Theconjugation reaction may be performed either with the RNA still bound tothe solid support or following cleavage of the RNA, in solution phase.Purification of the RNA conjugate by HPLC typically affords the pureconjugate.

Delivery of iRNA

The delivery of one or more iRNA to a subject in need thereof can beachieved in a number of different ways. In vivo delivery can beperformed directly by administering a composition comprising an iRNA,e.g. a dsRNA, to a subject. Alternatively, delivery can be performedindirectly by administering one or more vectors that encode and directthe expression of the iRNA. These alternatives are discussed furtherbelow.

Direct Delivery

In general, any method of delivering a nucleic acid molecule can beadapted for use with an iRNA (see e.g., Akhtar S, and Julian R L. (1992)Trends Cell. Biol. 2(5):139-144 and WO94/02595, which are incorporatedherein by reference in their entireties). However, there are threefactors that are important to consider in order to successfully deliveran iRNA molecule in vivo: (a) biological stability of the deliveredmolecule, (2) preventing non-specific effects, and (3) accumulation ofthe delivered molecule in the target tissue. The non-specific effects ofan iRNA can be minimized by local administration, for example by directinjection or implantation into a tissue (as a non-limiting example, atumor) or topically administering the preparation. Local administrationto a treatment site maximizes local concentration of the agent, limitsthe exposure of the agent to systemic tissues that may otherwise beharmed by the agent or that may degrade the agent, and permits a lowertotal dose of the iRNA molecule to be administered. Several studies haveshown successful knockdown of gene products when an iRNA is administeredlocally. For example, intraocular delivery of a VEGF dsRNA byintravitreal injection in cynomolgus monkeys (Tolentino, M J., et al(2004) Retina 24:132-138) and subretinal injections in mice (Reich, SJ., et al (2003) Mol. Vis. 9:210-216) were both shown to preventneovascularization in an experimental model of age-related maculardegeneration. In addition, direct intratumoral injection of a dsRNA inmice reduces tumor volume (Pille, J., et al (2005) Mol. Ther.11:267-274) and can prolong survival of tumor-bearing mice (Kim, W J.,et al (2006) Mol. Ther. 14:343-350; Li, S., et al (2007) Mol. Ther.15:515-523). RNA interference has also shown success with local deliveryto the CNS by direct injection (Dorn, G., et al. (2004) Nucleic Acids32:e49; Tan, P H., et al (2005) Gene Ther. 12:59-66; Makimura, H., et al(2002) BMC Neurosci. 3:18; Shishkina, G T., et al (2004) Neuroscience129:521-528; Thakker, E R., et al (2004) Proc. Natl. Acad. Sci. U.S.A.101:17270-17275; Akaneya, Y., et al (2005) J. Neurophysiol. 93:594-602)and to the lungs by intranasal administration (Howard, K A., et al(2006) Mol. Ther. 14:476-484; Zhang, X., et al (2004) J. Biol. Chem.279:10677-10684; Bitko, V., et al (2005) Nat. Med. 11:50-55). Foradministering an iRNA systemically for the treatment of a disease, theRNA can be modified or alternatively delivered using a drug deliverysystem; both methods act to prevent the rapid degradation of the dsRNAby endo- and exo-nucleases in vivo. Modification of the RNA or thepharmaceutical carrier can also permit targeting of the iRNA compositionto the target tissue and avoid undesirable off-target effects. iRNAmolecules can be modified by chemical conjugation to lipophilic groupssuch as cholesterol to enhance cellular uptake and prevent degradation.For example, an iRNA directed against ApoB conjugated to a lipophiliccholesterol moiety was injected systemically into mice and resulted inknockdown of apoB mRNA in both the liver and jejunum (Soutschek, J., etal (2004) Nature 432:173-178). Conjugation of an iRNA to an aptamer hasbeen shown to inhibit tumor growth and mediate tumor regression in amouse model of prostate cancer (McNamara, JO., et al (2006) Nat.Biotechnol. 24:1005-1015). In an alternative embodiment, the iRNA can bedelivered using drug delivery systems such as a nanoparticle, adendrimer, a polymer, liposomes, or a cationic delivery system.Positively charged cationic delivery systems facilitate binding of aniRNA molecule (negatively charged) and also enhance interactions at thenegatively charged cell membrane to permit efficient uptake of an iRNAby the cell. Cationic lipids, dendrimers, or polymers can either bebound to an iRNA, or induced to form a vesicle or micelle (see e.g., KimS H., et al (2008) Journal of Controlled Release 129(2):107-116) thatencases an iRNA. The formation of vesicles or micelles further preventsdegradation of the iRNA when administered systemically. Methods formaking and administering cationic-iRNA complexes are well within theabilities of one skilled in the art (see e.g., Sorensen, D R., et al(2003) J. Mol. Biol. 327:761-766; Verma, U N., et al (2003) Clin. CancerRes. 9:1291-1300; Arnold, A S et al (2007) J. Hypertens. 25:197-205,which are incorporated herein by reference in their entirety). Somenon-limiting examples of drug delivery systems useful for systemicdelivery of iRNAs include DOTAP (Sorensen, D R., et al (2003), supra;Verma, UN., et al (2003), supra), Oligofectamine, “solid nucleic acidlipid particles” (Zimmermann, T S., et al (2006) Nature 441:111-114),cardiolipin (Chien, P Y., et al (2005) Cancer Gene Ther. 12:321-328;Pal, A., et al (2005) Int J. Oncol. 26:1087-1091), polyethyleneimine(Bonnet M E., et al (2008) Pharm. Res. August 16 Epub ahead of print;Aigner, A. (2006) J. Biomed. Biotechnol. 71659), Arg-Gly-Asp (RGD)peptides (Liu, S. (2006) Mol. Pharm. 3:472-487), and polyamidoamines(Tomalia, D A., et al (2007) Biochem. Soc. Trans. 35:61-67; Yoo, H., etal (1999) Pharm. Res. 16:1799-1804). In some embodiments, an iRNA formsa complex with cyclodextrin for systemic administration. Methods foradministration and pharmaceutical compositions of iRNAs andcyclodextrins can be found in U.S. Pat. No. 7,427,605, which is hereinincorporated by reference in its entirety.

Vector Encoded dsRNAs

In another aspect, iRNA targeting one or more of the EGLN genes can beexpressed from transcription units inserted into DNA or RNA vectors(see, e.g., Couture, A, et al., TIG. (1996), 12:5-10; Skillern, A., etal., International PCT Publication No. WO 00/22113, Conrad,International PCT Publication No. WO 00/22114, and Conrad, U.S. Pat. No.6,054,299). Expression can be transient (on the order of hours to weeks)or sustained (weeks to months or longer), depending upon the specificconstruct used and the target tissue or cell type. These transgenes canbe introduced as a linear construct, a circular plasmid, or a viralvector, which can be an integrating or non-integrating vector. Thetransgene can also be constructed to permit it to be inherited as anextrachromosomal plasmid (Gassmann, et al., Proc. Natl. Acad. Sci. USA(1995) 92:1292).

The individual strand or strands of an iRNA can be transcribed from apromoter on an expression vector. Where two separate strands are to beexpressed to generate, for example, a dsRNA, two separate expressionvectors can be co-introduced (e.g., by transfection or infection) into atarget cell. Alternatively each individual strand of a dsRNA can betranscribed by promoters both of which are located on the sameexpression plasmid. In one embodiment, a dsRNA is expressed as aninverted repeat joined by a linker polynucleotide sequence such that thedsRNA has a stem and loop structure.

iRNA expression vectors are generally DNA plasmids or viral vectors.Expression vectors compatible with eukaryotic cells, preferably thosecompatible with vertebrate cells, can be used to produce recombinantconstructs for the expression of an iRNA as described herein. Eukaryoticcell expression vectors are well known in the art and are available froma number of commercial sources. Typically, such vectors are providedcontaining convenient restriction sites for insertion of the desirednucleic acid segment. Delivery of iRNA expressing vectors can besystemic, such as by intravenous or intramuscular administration, byadministration to target cells ex-planted from the patient followed byreintroduction into the patient, or by any other means that allows forintroduction into a desired target cell.

iRNA expression plasmids can be transfected into target cells as acomplex with cationic lipid carriers (e.g., Oligofectamine) ornon-cationic lipid-based carriers (e.g., Transit-TKO™). Multiple lipidtransfections for iRNA-mediated knockdowns targeting different regionsof a target RNA over a period of a week or more are also contemplated bythe invention. Successful introduction of vectors into host cells can bemonitored using various known methods. For example, transienttransfection can be signaled with a reporter, such as a fluorescentmarker, such as Green Fluorescent Protein (GFP). Stable transfection ofcells ex vivo can be ensured using markers that provide the transfectedcell with resistance to specific environmental factors (e.g.,antibiotics and drugs), such as hygromycin B resistance.

Viral vector systems which can be utilized with the methods andcompositions described herein include, but are not limited to, (a)adenovirus vectors; (b) retrovirus vectors, including but not limited tolentiviral vectors, moloney murine leukemia virus, etc.; (c)adeno-associated virus vectors; (d) herpes simplex virus vectors; (e) SV40 vectors; (f) polyoma virus vectors; (g) papilloma virus vectors; (h)picornavirus vectors; (i) pox virus vectors such as an orthopox, e.g.,vaccinia virus vectors or avipox, e.g. canary pox or fowl pox; and (j) ahelper-dependent or gutless adenovirus. Replication-defective virusescan also be advantageous. Different vectors will or will not becomeincorporated into the cells' genome. The constructs can include viralsequences for transfection, if desired. Alternatively, the construct maybe incorporated into vectors capable of episomal replication, e.g. EPVand EBV vectors. Constructs for the recombinant expression of an iRNAwill generally require regulatory elements, e.g., promoters, enhancers,etc., to ensure the expression of the iRNA in target cells. Otheraspects to consider for vectors and constructs are further describedbelow.

Vectors useful for the delivery of an iRNA will include regulatoryelements (promoter, enhancer, etc.) sufficient for expression of theiRNA in the desired target cell or tissue. The regulatory elements canbe chosen to provide either constitutive or regulated/inducibleexpression.

Expression of the iRNA can be precisely regulated, for example, by usingan inducible regulatory sequence that is sensitive to certainphysiological regulators, e.g., circulating glucose levels, or hormones(Docherty et al., 1994, FASEB J. 8:20-24). Such inducible expressionsystems, suitable for the control of dsRNA expression in cells or inmammals include, for example, regulation by ecdysone, by estrogen,progesterone, tetracycline, chemical inducers of dimerization, andisopropyl-beta-D1-thiogalactopyranoside (IPTG). A person skilled in theart would be able to choose the appropriate regulatory/promoter sequencebased on the intended use of the iRNA transgene.

In a specific embodiment, viral vectors that contain nucleic acidsequences encoding an iRNA can be used. For example, a retroviral vectorcan be used (see Miller et al., Meth. Enzymol. 217:581-599 (1993)).These retroviral vectors contain the components necessary for thecorrect packaging of the viral genome and integration into the host cellDNA. The nucleic acid sequences encoding an iRNA are cloned into one ormore vectors, which facilitates delivery of the nucleic acid into apatient. More detail about retroviral vectors can be found, for example,in Boesen et al., Biotherapy 6:291-302 (1994), which describes the useof a retroviral vector to deliver the mdr1 gene to hematopoietic stemcells in order to make the stem cells more resistant to chemotherapy.Other references illustrating the use of retroviral vectors in genetherapy are: Clowes et al., J. Clin. Invest. 93:644-651 (1994); Kiem etal., Blood 83:1467-1473 (1994); Salmons and Gunzberg, Human Gene Therapy4:129-141 (1993); and Grossman and Wilson, Curr. Opin. in Genetics andDevel. 3:110-114 (1993). Lentiviral vectors contemplated for useinclude, for example, the HIV based vectors described in U.S. Pat. Nos.6,143,520; 5,665,557; and 5,981,276, which are herein incorporated byreference.

Adenoviruses are also contemplated for use in delivery of iRNAs.Adenoviruses are especially attractive vehicles, e.g., for deliveringgenes to respiratory epithelia. Adenoviruses naturally infectrespiratory epithelia where they cause a mild disease. Other targets foradenovirus-based delivery systems are liver, the central nervous system,endothelial cells, and muscle. Adenoviruses have the advantage of beingcapable of infecting non-dividing cells. Kozarsky and Wilson, CurrentOpinion in Genetics and Development 3:499-503 (1993) present a review ofadenovirus-based gene therapy. Bout et al., Human Gene Therapy 5:3-10(1994) demonstrated the use of adenovirus vectors to transfer genes tothe respiratory epithelia of rhesus monkeys. Other instances of the useof adenoviruses in gene therapy can be found in Rosenfeld et al.,Science 252:431-434 (1991); Rosenfeld et al., Cell 68:143-155 (1992);Mastrangeli et al., J. Clin. Invest. 91:225-234 (1993); PCT PublicationWO94/12649; and Wang, et al., Gene Therapy 2:775-783 (1995). A suitableAV vector for expressing an iRNA featured in the invention, a method forconstructing the recombinant AV vector, and a method for delivering thevector into target cells, are described in Xia H et al. (2002), Nat.Biotech. 20: 1006-1010.

Use of Adeno-associated virus (AAV) vectors is also contemplated (Walshet al., Proc. Soc. Exp. Biol. Med. 204:289-300 (1993); U.S. Pat. No.5,436,146). In one embodiment, the iRNA can be expressed as twoseparate, complementary single-stranded RNA molecules from a recombinantAAV vector having, for example, either the U6 or H1 RNA promoters, orthe cytomegalovirus (CMV) promoter. Suitable AAV vectors for expressingthe dsRNA featured in the invention, methods for constructing therecombinant AV vector, and methods for delivering the vectors intotarget cells are described in Samulski R et al. (1987), J. Virol. 61:3096-3101; Fisher K J et al. (1996), J. Virol, 70: 520-532; Samulski Ret al. (1989), J. Virol. 63: 3822-3826; U.S. Pat. No. 5,252,479; U.S.Pat. No. 5,139,941; International Patent Application No. WO 94/13788;and International Patent Application No. WO 93/24641, the entiredisclosures of which are herein incorporated by reference.

Another preferred viral vector is a pox virus such as a vaccinia virus,for example an attenuated vaccinia such as Modified Virus Ankara (MVA)or NYVAC, an avipox such as fowl pox or canary pox.

The tropism of viral vectors can be modified by pseudotyping the vectorswith envelope proteins or other surface antigens from other viruses, orby substituting different viral capsid proteins, as appropriate. Forexample, lentiviral vectors can be pseudotyped with surface proteinsfrom vesicular stomatitis virus (VSV), rabies, Ebola, Mokola, and thelike. AAV vectors can be made to target different cells by engineeringthe vectors to express different capsid protein serotypes; see, e.g.,Rabinowitz J E et al. (2002), J Virol 76:791-801, the entire disclosureof which is herein incorporated by reference.

The pharmaceutical preparation of a vector can include the vector in anacceptable diluent, or can include a slow release matrix in which thegene delivery vehicle is imbedded. Alternatively, where the completegene delivery vector can be produced intact from recombinant cells,e.g., retroviral vectors, the pharmaceutical preparation can include oneor more cells which produce the gene delivery system.

III. PHARMACEUTICAL COMPOSITIONS CONTAINING iRNA

In one embodiment, the invention provides pharmaceutical compositionscontaining an iRNA, as described herein, and a pharmaceuticallyacceptable carrier. The pharmaceutical composition containing the iRNAis useful for treating a disease or disorder associated with theexpression or activity of an EGLN gene, such as pathological processesmediated by EGLN expression. Such pharmaceutical compositions areformulated based on the mode of delivery. One example is compositionsthat are formulated for systemic administration via parenteral delivery,e.g., by intravenous (IV) delivery. Another example is compositions thatare formulated for direct delivery into the brain parenchyma, e.g., byinfusion into the brain, such as by continuous pump infusion.

The pharmaceutical compositions featured herein are administered indosages sufficient to inhibit expression of EGLN genes. In general, asuitable dose of iRNA will be in the range of 0.01 to 200.0 milligramsper kilogram body weight of the recipient per day, generally in therange of 1 to 50 mg per kilogram body weight per day. For example, thedsRNA can be administered at 0.05 mg/kg, 0.5 mg/kg, 1 mg/kg, 1.5 mg/kg,2 mg/kg, 3 mg/kg, 10 mg/kg, 20 mg/kg, 30 mg/kg, 40 mg/kg, or 50 mg/kgper single dose. The pharmaceutical composition may be administered oncedaily, or the iRNA may be administered as two, three, or more sub-dosesat appropriate intervals throughout the day or even using continuousinfusion or delivery through a controlled release formulation. In thatcase, the iRNA contained in each sub-dose must be correspondinglysmaller in order to achieve the total daily dosage. The dosage unit canalso be compounded for delivery over several days, e.g., using aconventional sustained release formulation which provides sustainedrelease of the iRNA over a several day period. Sustained releaseformulations are well known in the art and are particularly useful fordelivery of agents at a particular site, such as could be used with theagents of the present invention. In this embodiment, the dosage unitcontains a corresponding multiple of the daily dose.

The effect of a single dose on EGLN levels can be long lasting, suchthat subsequent doses are administered at not more than 3, 4, or 5 dayintervals, or at not more than 1, 2, 3, or 4 week intervals.

The skilled artisan will appreciate that certain factors may influencethe dosage and timing required to effectively treat a subject, includingbut not limited to the severity of the disease or disorder, previoustreatments, the general health and/or age of the subject, and otherdiseases present. Moreover, treatment of a subject with atherapeutically effective amount of a composition can include a singletreatment or a series of treatments. Estimates of effective dosages andin vivo half-lives for the individual iRNAs encompassed by the inventioncan be made using conventional methodologies or on the basis of in vivotesting using an appropriate animal model, as described elsewhereherein.

Advances in mouse genetics have generated a number of mouse models forthe study of various human diseases, such as pathological processesmediated by EGLN expression. Such models can be used for in vivo testingof iRNA, as well as for determining a therapeutically effective dose. Asuitable mouse model is, for example, a mouse containing a transgeneexpressing human EGLN.

The present invention also includes pharmaceutical compositions andformulations that include the iRNA compounds featured in the invention.The pharmaceutical compositions of the present invention may beadministered in a number of ways depending upon whether local orsystemic treatment is desired and upon the area to be treated.Administration may be topical (e.g., by a transdermal patch), pulmonary,e.g., by inhalation or insufflation of powders or aerosols, including bynebulizer; intratracheal, intranasal, epidermal and transdermal, oral orparenteral. Parenteral administration includes intravenous,intraarterial, subcutaneous, intraperitoneal or intramuscular injectionor infusion; subdermal, e.g., via an implanted device; or intracranial,e.g., by intraparenchymal, intrathecal or intraventricular,administration.

The iRNA can be delivered in a manner to target a particular tissue,such as the liver (e.g., the hepatocytes of the liver).

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable. Coated condoms, gloves and thelike may also be useful. Suitable topical formulations include those inwhich the iRNAs featured in the invention are in admixture with atopical delivery agent such as lipids, liposomes, fatty acids, fattyacid esters, steroids, chelating agents and surfactants. Suitable lipidsand liposomes include neutral (e.g., dioleoylphosphatidyl DOPEethanolamine, dimyristoylphosphatidyl choline DMPC,distearolyphosphatidyl choline) negative (e.g., dimyristoylphosphatidylglycerol DMPG) and cationic (e.g., dioleoyltetramethylaminopropyl DOTAPand dioleoylphosphatidyl ethanolamine DOTMA). iRNAs featured in theinvention may be encapsulated within liposomes or may form complexesthereto, in particular to cationic liposomes. Alternatively, iRNAs maybe complexed to lipids, in particular to cationic lipids. Suitable fattyacids and esters include but are not limited to arachidonic acid, oleicacid, eicosanoic acid, lauric acid, caprylic acid, capric acid, myristicacid, palmitic acid, stearic acid, linoleic acid, linolenic acid,dicaprate, tricaprate, monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or aC₁₋₂₀ alkyl ester (e.g., isopropylmyristate IPM), monoglyceride,diglyceride or pharmaceutically acceptable salt thereof. Topicalformulations are described in detail in U.S. Pat. No. 6,747,014, whichis incorporated herein by reference.

Liposomal Formulations

There are many organized surfactant structures besides microemulsionsthat have been studied and used for the formulation of drugs. Theseinclude monolayers, micelles, bilayers and vesicles. Vesicles, such asliposomes, have attracted great interest because of their specificityand the duration of action they offer from the standpoint of drugdelivery. As used in the present invention, the term “liposome” means avesicle composed of amphiphilic lipids arranged in a spherical bilayeror bilayers.

Liposomes are unilamellar or multilamellar vesicles which have amembrane formed from a lipophilic material and an aqueous interior. Theaqueous portion contains the composition to be delivered. Cationicliposomes possess the advantage of being able to fuse to the cell wall.Non-cationic liposomes, although not able to fuse as efficiently withthe cell wall, are taken up by macrophages in vivo.

In order to traverse intact mammalian skin, lipid vesicles must passthrough a series of fine pores, each with a diameter less than 50 nm,under the influence of a suitable transdermal gradient. Therefore, it isdesirable to use a liposome which is highly deformable and able to passthrough such fine pores.

Further advantages of liposomes include; liposomes obtained from naturalphospholipids are biocompatible and biodegradable; liposomes canincorporate a wide range of water and lipid soluble drugs; liposomes canprotect encapsulated drugs in their internal compartments frommetabolism and degradation (Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245). Important considerations in thepreparation of liposome formulations are the lipid surface charge,vesicle size and the aqueous volume of the liposomes.

Liposomes are useful for the transfer and delivery of active ingredientsto the site of action. Because the liposomal membrane is structurallysimilar to biological membranes, when liposomes are applied to a tissue,the liposomes start to merge with the cellular membranes and as themerging of the liposome and cell progresses, the liposomal contents areemptied into the cell where the active agent may act.

Liposomal formulations have been the focus of extensive investigation asthe mode of delivery for many drugs. There is growing evidence that fortopical administration, liposomes present several advantages over otherformulations. Such advantages include reduced side-effects related tohigh systemic absorption of the administered drug, increasedaccumulation of the administered drug at the desired target, and theability to administer a wide variety of drugs, both hydrophilic andhydrophobic, into the skin.

Several reports have detailed the ability of liposomes to deliver agentsincluding high-molecular weight DNA into the skin. Compounds includinganalgesics, antibodies, hormones and high-molecular weight DNAs havebeen administered to the skin. The majority of applications resulted inthe targeting of the upper epidermis

Liposomes fall into two broad classes. Cationic liposomes are positivelycharged liposomes which interact with the negatively charged DNAmolecules to form a stable complex. The positively charged DNA/liposomecomplex binds to the negatively charged cell surface and is internalizedin an endosome. Due to the acidic pH within the endosome, the liposomesare ruptured, releasing their contents into the cell cytoplasm (Wang etal., Biochem. Biophys. Res. Commun., 1987, 147, 980-985).

Liposomes which are pH-sensitive or negatively-charged, entrap DNArather than complex with it. Since both the DNA and the lipid aresimilarly charged, repulsion rather than complex formation occurs.Nevertheless, some DNA is entrapped within the aqueous interior of theseliposomes. pH-sensitive liposomes have been used to deliver DNA encodingthe thymidine kinase gene to cell monolayers in culture. Expression ofthe exogenous gene was detected in the target cells (Zhou et al.,Journal of Controlled Release, 1992, 19, 269-274).

One major type of liposomal composition includes phospholipids otherthan naturally-derived phosphatidylcholine. Neutral liposomecompositions, for example, can be formed from dimyristoylphosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC).Anionic liposome compositions generally are formed from dimyristoylphosphatidylglycerol, while anionic fusogenic liposomes are formedprimarily from dioleoyl phosphatidylethanolamine (DOPE). Another type ofliposomal composition is formed from phosphatidylcholine (PC) such as,for example, soybean PC, and egg PC. Another type is formed frommixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

Several studies have assessed the topical delivery of liposomal drugformulations to the skin. Application of liposomes containing interferonto guinea pig skin resulted in a reduction of skin herpes sores whiledelivery of interferon via other means (e.g., as a solution or as anemulsion) were ineffective (Weiner et al., Journal of Drug Targeting,1992, 2, 405-410). Further, an additional study tested the efficacy ofinterferon administered as part of a liposomal formulation to theadministration of interferon using an aqueous system, and concluded thatthe liposomal formulation was superior to aqueous administration (duPlessis et al., Antiviral Research, 1992, 18, 259-265).

Non-ionic liposomal systems have also been examined to determine theirutility in the delivery of drugs to the skin, in particular systemscomprising non-ionic surfactant and cholesterol. Non-ionic liposomalformulations comprising Novasome™ I (glyceryldilaurate/cholesterol/polyoxyethylene-10-stearyl ether) and Novasome™ II(glyceryl distearate/cholesterol/polyoxyethylene-10-stearyl ether) wereused to deliver cyclosporin-A into the dermis of mouse skin. Resultsindicated that such non-ionic liposomal systems were effective infacilitating the deposition of cyclosporin-A into different layers ofthe skin (Hu et al. S.T.P.Pharma. Sci., 1994, 4, 6, 466).

Liposomes also include “sterically stabilized” liposomes, a term which,as used herein, refers to liposomes comprising one or more specializedlipids that, when incorporated into liposomes, result in enhancedcirculation lifetimes relative to liposomes lacking such specializedlipids. Examples of sterically stabilized liposomes are those in whichpart of the vesicle-forming lipid portion of the liposome (A) comprisesone or more glycolipids, such as monosialoganglioside G_(M1), or (B) isderivatized with one or more hydrophilic polymers, such as apolyethylene glycol (PEG) moiety. While not wishing to be bound by anyparticular theory, it is thought in the art that, at least forsterically stabilized liposomes containing gangliosides, sphingomyelin,or PEG-derivatized lipids, the enhanced circulation half-life of thesesterically stabilized liposomes derives from a reduced uptake into cellsof the reticuloendothelial system (RES) (Allen et al., FEBS Letters,1987, 223, 42; Wu et al., Cancer Research, 1993, 53, 3765).

Various liposomes comprising one or more glycolipids are known in theart. Papahadjopoulos et al. (Ann. N.Y. Acad. Sci., 1987, 507, 64)reported the ability of monosialoganglioside G_(M1), galactocerebrosidesulfate and phosphatidylinositol to improve blood half-lives ofliposomes. These findings were expounded upon by Gabizon et al. (Proc.Natl. Acad. Sci. U.S.A., 1988, 85, 6949). U.S. Pat. No. 4,837,028 and WO88/04924, both to Allen et al., disclose liposomes comprising (1)sphingomyelin and (2) the ganglioside G_(M1) or a galactocerebrosidesulfate ester. U.S. Pat. No. 5,543,152 (Webb et al.) discloses liposomescomprising sphingomyelin. Liposomes comprising1,2-sn-dimyristoylphosphatidylcholine are disclosed in WO 97/13499 (Limet al).

Many liposomes comprising lipids derivatized with one or morehydrophilic polymers, and methods of preparation thereof, are known inthe art. Sunamoto et al. (Bull. Chem. Soc. Jpn., 1980, 53, 2778)described liposomes comprising a nonionic detergent, 2C_(1215G), thatcontains a PEG moiety. Illum et al. (FEBS Lett., 1984, 167, 79) notedthat hydrophilic coating of polystyrene particles with polymeric glycolsresults in significantly enhanced blood half-lives. Syntheticphospholipids modified by the attachment of carboxylic groups ofpolyalkylene glycols (e.g., PEG) are described by Sears (U.S. Pat. Nos.4,426,330 and 4,534,899). Klibanov et al. (FEBS Lett., 1990, 268, 235)described experiments demonstrating that liposomes comprisingphosphatidylethanolamine (PE) derivatized with PEG or PEG stearate havesignificant increases in blood circulation half-lives. Blume et al.(Biochimica et Biophysica Acta, 1990, 1029, 91) extended suchobservations to other PEG-derivatized phospholipids, e.g., DSPE-PEG,formed from the combination of distearoylphosphatidylethanolamine (DSPE)and PEG. Liposomes having covalently bound PEG moieties on theirexternal surface are described in European Patent No. EP 0 445 131 B1and WO 90/04384 to Fisher. Liposome compositions containing 1-20 molepercent of PE derivatized with PEG, and methods of use thereof, aredescribed by Woodle et al. (U.S. Pat. Nos. 5,013,556 and 5,356,633) andMartin et al. (U.S. Pat. No. 5,213,804 and European Patent No. EP 0 496813 B1). Liposomes comprising a number of other lipid-polymer conjugatesare disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 (both to Martinet al.) and in WO 94/20073 (Zalipsky et al.) Liposomes comprisingPEG-modified ceramide lipids are described in WO 96/10391 (Choi et al).U.S. Pat. No. 5,540,935 (Miyazaki et al.) and U.S. Pat. No. 5,556,948(Tagawa et al.) describe PEG-containing liposomes that can be furtherderivatized with functional moieties on their surfaces.

A number of liposomes comprising nucleic acids are known in the art. WO96/40062 to Thierry et al. discloses methods for encapsulating highmolecular weight nucleic acids in liposomes. U.S. Pat. No. 5,264,221 toTagawa et al. discloses protein-bonded liposomes and asserts that thecontents of such liposomes may include a dsRNA. U.S. Pat. No. 5,665,710to Rahman et al. describes certain methods of encapsulatingoligodeoxynucleotides in liposomes. WO 97/04787 to Love et al. disclosesliposomes comprising dsRNAs targeted to the raf gene.

Transfersomes are yet another type of liposomes, and are highlydeformable lipid aggregates which are attractive candidates for drugdelivery vehicles. Transfersomes may be described as lipid dropletswhich are so highly deformable that they are easily able to penetratethrough pores which are smaller than the droplet. Transfersomes areadaptable to the environment in which they are used, e.g., they areself-optimizing (adaptive to the shape of pores in the skin),self-repairing, frequently reach their targets without fragmenting, andoften self-loading. To make transfersomes it is possible to add surfaceedge-activators, usually surfactants, to a standard liposomalcomposition. Transfersomes have been used to deliver serum albumin tothe skin. The transfersome-mediated delivery of serum albumin has beenshown to be as effective as subcutaneous injection of a solutioncontaining serum albumin.

Surfactants find wide application in formulations such as emulsions(including microemulsions) and liposomes. The most common way ofclassifying and ranking the properties of the many different types ofsurfactants, both natural and synthetic, is by the use of thehydrophile/lipophile balance (HLB). The nature of the hydrophilic group(also known as the “head”) provides the most useful means forcategorizing the different surfactants used in formulations (Rieger, inPharmaceutical Dosage Forms, Marcel Dekker, Inc., New York, N.Y., 1988,p. 285).

If the surfactant molecule is not ionized, it is classified as anonionic surfactant. Nonionic surfactants find wide application inpharmaceutical and cosmetic products and are usable over a wide range ofpH values. In general their HLB values range from 2 to about 18depending on their structure. Nonionic surfactants include nonionicesters such as ethylene glycol esters, propylene glycol esters, glycerylesters, polyglyceryl esters, sorbitan esters, sucrose esters, andethoxylated esters. Nonionic alkanolamides and ethers such as fattyalcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylatedblock polymers are also included in this class. The polyoxyethylenesurfactants are the most popular members of the nonionic surfactantclass.

If the surfactant molecule carries a negative charge when it isdissolved or dispersed in water, the surfactant is classified asanionic. Anionic surfactants include carboxylates such as soaps, acyllactylates, acyl amides of amino acids, esters of sulfuric acid such asalkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkylbenzene sulfonates, acyl isethionates, acyl taurates andsulfosuccinates, and phosphates. The most important members of theanionic surfactant class are the alkyl sulfates and the soaps.

If the surfactant molecule carries a positive charge when it isdissolved or dispersed in water, the surfactant is classified ascationic. Cationic surfactants include quaternary ammonium salts andethoxylated amines. The quaternary ammonium salts are the most usedmembers of this class.

If the surfactant molecule has the ability to carry either a positive ornegative charge, the surfactant is classified as amphoteric. Amphotericsurfactants include acrylic acid derivatives, substituted alkylamides,N-alkylbetaines and phosphatides.

The use of surfactants in drug products, formulations and in emulsionshas been reviewed (Rieger, in Pharmaceutical Dosage Forms, MarcelDekker, Inc., New York, N.Y., 1988, p. 285).

Nucleic Acid Lipid Particles

In one embodiment, an EGLN dsRNA featured in the invention is fullyencapsulated in the lipid formulation, e.g., to form a SPLP, pSPLP,SNALP, or other nucleic acid-lipid particle. As used herein, the term“SNALP” refers to a stable nucleic acid-lipid particle, including SPLP.As used herein, the term “SPLP” refers to a nucleic acid-lipid particlecomprising plasmid DNA encapsulated within a lipid vesicle. SNALPs andSPLPs typically contain a cationic lipid, a non-cationic lipid, and alipid that prevents aggregation of the particle (e.g., a PEG-lipidconjugate). SNALPs and SPLPs are extremely useful for systemicapplications, as they exhibit extended circulation lifetimes followingintravenous (i.v.) injection and accumulate at distal sites (e.g., sitesphysically separated from the administration site). SPLPs include“pSPLP,” which include an encapsulated condensing agent-nucleic acidcomplex as set forth in PCT Publication No. WO 00/03683. The particlesof the present invention typically have a mean diameter of about 50 nmto about 150 nm, more typically about 60 nm to about 130 nm, moretypically about 70 nm to about 110 nm, most typically about 70 nm toabout 90 nm, and are substantially nontoxic. In addition, the nucleicacids when present in the nucleic acid-lipid particles of the presentinvention are resistant in aqueous solution to degradation with anuclease. Nucleic acid-lipid particles and their method of preparationare disclosed in, e.g., U.S. Pat. Nos. 5,976,567; 5,981,501; 6,534,484;6,586,410; 6,815,432; and PCT Publication No. WO 96/40964.

In one embodiment, the lipid to drug ratio (mass/mass ratio) (e.g.,lipid to dsRNA ratio) will be in the range of from about 1:1 to about50:1, from about 1:1 to about 25:1, from about 3:1 to about 15:1, fromabout 4:1 to about 10:1, from about 5:1 to about 9:1, or about 6:1 toabout 9:1.

The cationic lipid may be, for example, N,N-dioleyl-N,N-dimethylammoniumchloride (DODAC), N,N-distearyl-N,N-dimethylammonium bromide (DDAB),N-(1-(2,3-dioleoyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTAP),N-(1-(2,3-dioleyloxy)propyl)-N,N,N-trimethylammonium chloride (DOTMA),N,N-dimethyl-2,3-dioleyloxy)propylamine (DODMA),1,2-DiLinoleyloxy-N,N-dimethylaminopropane (DLinDMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLenDMA),1,2-Dilinoleylcarbamoyloxy-3-dimethylaminopropane (DLin-C-DAP),1,2-Dilinoleyoxy-3-(dimethylamino)acetoxypropane (DLin-DAC),1,2-Dilinoleyoxy-3-morpholinopropane (DLin-MA),1,2-Dilinoleoyl-3-dimethylaminopropane (DLinDAP),1,2-Dilinoleylthio-3-dimethylaminopropane (DLin-S-DMA),1-Linoleoyl-2-linoleyloxy-3-dimethylaminopropane (DLin-2-DMAP),1,2-Dilinoleyloxy-3-trimethylaminopropane chloride salt (DLin-TMA.Cl),1,2-Dilinoleoyl-3-trimethylaminopropane chloride salt (DLin-TAP.Cl),1,2-Dilinoleyloxy-3-(N-methylpiperazino)propane (DLin-MPZ), or3-(N,N-Dilinoleylamino)-1,2-propanediol (DLinAP),3-(N,N-Dioleylamino)-1,2-propanedio (DOAP),1,2-Dilinoleyloxo-3-(2-N,N-dimethylamino)ethoxypropane (DLin-EG-DMA),1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA),2,2-Dilinoleyl-4-dimethylaminomethyl-[1,3]-dioxolane (DLin-K-DMA) oranalogs thereof,(3aR,5s,6aS)—N,N-dimethyl-2,2-di((9Z,12Z)-octadeca-9,12-dienyl)tetrahydro-3aH-cyclopenta[d][1,3]dioxol-5-amine(ALN100), (6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate (MC3),1,1′-(2-(4-(2-((2-(bis(2-hydroxydodecyl)amino)ethyl)(2-hydroxydodecyl)amino)ethyl)piperazin-1-yl)ethylazanediyl)didodecan-2-ol(Tech G1), or a mixture thereof. The cationic lipid may comprise fromabout 20 mol % to about 50 mol % or about 40 mol % of the total lipidpresent in the particle.

In another embodiment, the compound2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane can be used toprepare lipid-siRNA nanoparticles. Synthesis of2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane is described in U.S.provisional patent application No. 61/107,998 filed on Oct. 23, 2008,which is herein incorporated by reference.

In one embodiment, the lipid-siRNA particle includes 40%2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane: 10% DSPC: 40%Cholesterol: 10% PEG-C-DOMG (mole percent) with a particle size of63.0±20 nm and a 0.027 siRNA/Lipid Ratio.

The non-cationic lipid may be an anionic lipid or a neutral lipidincluding, but not limited to, distearoylphosphatidylcholine (DSPC),dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine(DPPC), dioleoylphosphatidylglycerol (DOPG),dipalmitoylphosphatidylglycerol (DPPG),dioleoyl-phosphatidylethanolamine (DOPE),palmitoyloleoylphosphatidylcholine (POPC),palmitoyloleoylphosphatidylethanolamine (POPE),dioleoyl-phosphatidylethanolamine4-(N-maleimidomethyl)-cyclohexane-1-carboxylate (DOPE-mal), dipalmitoylphosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE),distearoyl-phosphatidyl-ethanolamine (DSPE), 16-O-monomethyl PE,16-O-dimethyl PE, 18-1-trans PE,1-stearoyl-2-oleoyl-phosphatidyethanolamine (SOPE), cholesterol, or amixture thereof. The non-cationic lipid may be from about 5 mol % toabout 90 mol %, about 10 mol %, or about 58 mol % if cholesterol isincluded, of the total lipid present in the particle.

The conjugated lipid that inhibits aggregation of particles may be, forexample, a polyethyleneglycol (PEG)-lipid including, without limitation,a PEG-diacylglycerol (DAG), a PEG-dialkyloxypropyl (DAA), aPEG-phospholipid, a PEG-ceramide (Cer), or a mixture thereof. ThePEG-DAA conjugate may be, for example, a PEG-dilauryloxypropyl (Ci₂), aPEG-dimyristyloxypropyl (Ci₄), a PEG-dipalmityloxypropyl (Ci₆), or aPEG-distearyloxypropyl (C]₈). The conjugated lipid that preventsaggregation of particles may be from 0 mol % to about 20 mol % or about2 mol % of the total lipid present in the particle.

In some embodiments, the nucleic acid-lipid particle further includescholesterol at, e.g., about 10 mol % to about 60 mol % or about 48 mol %of the total lipid present in the particle.

LNP01

In one embodiment, the lipidoid ND98.4HCl (MW 1487) (see U.S. patentapplication Ser. No. 12/056,230, filed Mar. 26, 2008, which is hereinincorporated by reference), Cholesterol (Sigma-Aldrich), andPEG-Ceramide C16 (Avanti Polar Lipids) can be used to preparelipid-dsRNA nanoparticles (i.e., LNP01 particles). Stock solutions ofeach in ethanol can be prepared as follows: ND98, 133 mg/ml;Cholesterol, 25 mg/ml, PEG-Ceramide C16, 100 mg/ml. The ND98,Cholesterol, and PEG-Ceramide C16 stock solutions can then be combinedin a, e.g., 42:48:10 molar ratio. The combined lipid solution can bemixed with aqueous dsRNA (e.g., in sodium acetate pH 5) such that thefinal ethanol concentration is about 35-45% and the final sodium acetateconcentration is about 100-300 mM. Lipid-dsRNA nanoparticles typicallyform spontaneously upon mixing. Depending on the desired particle sizedistribution, the resultant nanoparticle mixture can be extruded througha polycarbonate membrane (e.g., 100 nm cut-off) using, for example, athermobarrel extruder, such as Lipex Extruder (Northern Lipids, Inc). Insome cases, the extrusion step can be omitted. Ethanol removal andsimultaneous buffer exchange can be accomplished by, for example,dialysis or tangential flow filtration. Buffer can be exchanged with,for example, phosphate buffered saline (PBS) at about pH 7, e.g., aboutpH 6.9, about pH 7.0, about pH 7.1, about pH 7.2, about pH 7.3, or aboutpH 7.4.

LNP01 formulations are described, e.g., in International ApplicationPublication No. WO 2008/042973, which is hereby incorporated byreference.

Additional exemplary lipid-dsRNA formulations are as follows:

cationic lipid/non-cationic lipid/cholesterol/PEG-lipid conjugateCationic Lipid Lipid:siRNA ratio SNALP 1,2-Dilinolenyloxy-N,N-DLinDMA/DPPC/Cholesterol/PEG- dimethylaminopropane (DLinDMA) cDMA(57.1/7.1/34.4/1.4) lipid:siRNA ~7:l S-XTC2,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DPPC/Cholesterol/PEG-cDMA[1,3]-dioxolane (XTC) 57.1/7.1/34.4/1.4 lipid:siRNA ~7:1 LNP052,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~6:1 LNP062,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 57.5/7.5/31.5/3.5 lipid:siRNA ~11:1 LNP072,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~6:1 LNP082,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 60/7.5/31/1.5, lipid:siRNA ~11:1 LNP092,2-Dilinoleyl-4-dimethylaminoethyl- XTC/DSPC/Cholesterol/PEG-DMG[1,3]-dioxolane (XTC) 50/10/38.5/1.5 Lipid:siRNA 10:1 LNP10(3aR,5s,6aS)-N,N-dimethyl-2,2- ALN100/DSPC/Cholesterol/PEG-DMGdi((9Z,12Z)-octadeca-9,12- 50/10/38.5/1.5 dienyl)tetrahydro-3aH-Lipid:siRNA 10:1 cyclopenta[d][1,3]-dioxol-5-amine (ALN100) LNP11(6Z,9Z,28Z,31Z)-heptatriaconta- MC-3/DSPC/Cholesterol/PEG-DMG6,9,28,31-tetraen-19-yl 4- 50/10/38.5/1.5 (dimethylamino)butanoate (MC3)Lipid:siRNA 10:1 LNP12 1,1′-(2-(4-(2-((2-(bis(2-C12-200/DSPC/Cholesterol/PEG-DMG hydroxydodecyl)amino)ethyl)(2-50/10/38.5/1.5 hydroxydodecyl)amino)ethyl)piperazin- Lipid:siRNA 10:11-yl)ethylazanediyl)didodecan-2-ol (C12-200) LNP13 XTCXTC/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 33:1 LNP14 MC3MC3/DSPC/Chol/PEG-DMG 40/15/40/5 Lipid:siRNA: 11:1 LNP15 MC3MC3/DSPC/Chol/PEG-DSG/GalNAc- PEG-DSG 50/10/35/4.5/0.5 Lipid:siRNA: 11:1LNP16 MC3 MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP17MC3 MC3/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP18 MC3MC3/DSPC/Chol/PEG-DMG 50/10/38.5/1.5 Lipid:siRNA: 12:1 LNP19 MC3MC3/DSPC/Chol/PEG-DMG 50/10/35/5 Lipid:siRNA: 8:1 LNP20 MC3MC3/DSPC/Chol/PEG-DPG 50/10/38.5/1.5 Lipid:siRNA: 10:1 LNP21 C12-200C12-200/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 7:1 LNP22 XTCXTC/DSPC/Chol/PEG-DSG 50/10/38.5/1.5 Lipid:siRNA: 10:1 DSPC:distearoylphosphatidylcholine DPPC: dipalmitoylphosphatidylcholinePEG-DMG: PEG-didimyristoyl glycerol (C14-PEG, or PEG-C14) (PEG with avgmol wt of 2000) PEG-DSG: PEG-distyryl glycerol (C18-PEG, or PEG-C18)(PEG with avg mol wt of 2000) PEG-cDMA:PEG-carbamoyl-1,2-dimyristyloxypropylamine (PEG with avg mol wt of 2000)

SNALP (1,2-Dilinolenyloxy-N,N-dimethylaminopropane (DLinDMA)) comprisingformulations are described in International Publication No.WO2009/127060, filed Apr. 15, 2009, which is hereby incorporated byreference.

XTC comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/148,366, filed Jan. 29, 2009; U.S. Provisional Ser. No.61/156,851, filed Mar. 2, 2009; U.S. Provisional Serial No. filed Jun.10, 2009; U.S. Provisional Ser. No. 61/228,373, filed Jul. 24, 2009;U.S. Provisional Ser. No. 61/239,686, filed Sep. 3, 2009, andInternational Application No. PCT/US2010/022614, filed Jan. 29, 2010,which are hereby incorporated by reference.

MC3 comprising formulations are described, e.g., in U.S. ProvisionalSer. No. 61/244,834, filed Sep. 22, 2009, U.S. Provisional Ser. No.61/185,800, filed Jun. 10, 2009, and International Application No.PCT/US10/28224, filed Jun. 10, 2010, which are hereby incorporated byreference.

ALNY-100 comprising formulations are described, e.g., Internationalpatent application number PCT/US09/63933, filed on Nov. 10, 2009, whichis hereby incorporated by reference.

C12-200 comprising formulations are described in U.S. Provisional Ser.No. 61/175,770, filed May 5, 2009 and International Application No.PCT/US10/33777, filed May 5, 2010, which are hereby incorporated byreference.

Synthesis of Cationic Lipids

Any of the compounds, e.g., cationic lipids and the like, used in thenucleic acid-lipid particles of the invention may be prepared by knownorganic synthesis techniques, including the methods described in moredetail in the Examples. All substituents are as defined below unlessindicated otherwise.

“Alkyl” means a straight chain or branched, noncyclic or cyclic,saturated aliphatic hydrocarbon containing from 1 to 24 carbon atoms.Representative saturated straight chain alkyls include methyl, ethyl,n-propyl, n-butyl, n-pentyl, n-hexyl, and the like; while saturatedbranched alkyls include isopropyl, sec-butyl, isobutyl, ten-butyl,isopentyl, and the like. Representative saturated cyclic alkyls includecyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and the like; whileunsaturated cyclic alkyls include cyclopentenyl and cyclohexenyl, andthe like.

“Alkenyl” means an alkyl, as defined above, containing at least onedouble bond between adjacent carbon atoms. Alkenyls include both cis andtrans isomers. Representative straight chain and branched alkenylsinclude ethylenyl, propylenyl, 1-butenyl, 2-butenyl, isobutylenyl,1-pentenyl, 2-pentenyl, 3-methyl-1-butenyl, 2-methyl-2-butenyl,2,3-dimethyl-2-butenyl, and the like.

“Alkynyl” means any alkyl or alkenyl, as defined above, whichadditionally contains at least one triple bond between adjacent carbons.Representative straight chain and branched alkynyls include acetylenyl,propynyl, 1-butynyl, 2-butynyl, 1-pentynyl, 2-pentynyl, 3-methyl-1butynyl, and the like.

“Acyl” means any alkyl, alkenyl, or alkynyl wherein the carbon at thepoint of attachment is substituted with an oxo group, as defined below.For example, —C(═O)alkyl, —C(═O)alkenyl, and —C(═O)alkynyl are acylgroups.

“Heterocycle” means a 5- to 7-membered monocyclic, or 7- to 10-memberedbicyclic, heterocyclic ring which is either saturated, unsaturated, oraromatic, and which contains from 1 or 2 heteroatoms independentlyselected from nitrogen, oxygen and sulfur, and wherein the nitrogen andsulfur heteroatoms may be optionally oxidized, and the nitrogenheteroatom may be optionally quaternized, including bicyclic rings inwhich any of the above heterocycles are fused to a benzene ring. Theheterocycle may be attached via any heteroatom or carbon atom.Heterocycles include heteroaryls as defined below. Heterocycles includemorpholinyl, pyrrolidinonyl, pyrrolidinyl, piperidinyl, piperizynyl,hydantoinyl, valerolactamyl, oxiranyl, oxetanyl, tetrahydrofuranyl,tetrahydropyranyl, tetrahydropyridinyl, tetrahydroprimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, tetrahydropyrimidinyl,tetrahydrothiophenyl, tetrahydrothiopyranyl, and the like.

The terms “optionally substituted alkyl”, “optionally substitutedalkenyl”, “optionally substituted alkynyl”, “optionally substitutedacyl”, and “optionally substituted heterocycle” means that, whensubstituted, at least one hydrogen atom is replaced with a substituent.In the case of an oxo substituent (═O) two hydrogen atoms are replaced.In this regard, substituents include oxo, halogen, heterocycle, —CN,—OR^(x), —NR^(x)R^(y), —NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x),—C(═O)OR^(x), —C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y),wherein n is 0, 1 or 2, R^(x) and R^(y) are the same or different andindependently hydrogen, alkyl or heterocycle, and each of said alkyl andheterocycle substituents may be further substituted with one or more ofoxo, halogen, —OH, —CN, alkyl, —OR^(x), heterocycle, —NR^(x)R^(y),—NR^(x)C(═O)R^(y), —NR^(x)SO₂R^(y), —C(═O)R^(x), —C(═O)OR^(x),—C(═O)NR^(x)R^(y), —SO_(n)R^(x) and —SO_(n)NR^(x)R^(y).

“Halogen” means fluoro, chloro, bromo and iodo.

In some embodiments, the methods of the invention may require the use ofprotecting groups. Protecting group methodology is well known to thoseskilled in the art (see, for example, PROTECTIVE GROUPS IN ORGANICSYNTHESIS, Green, T. W. et al., Wiley-Interscience, New York City,1999). Briefly, protecting groups within the context of this inventionare any group that reduces or eliminates unwanted reactivity of afunctional group. A protecting group can be added to a functional groupto mask its reactivity during certain reactions and then removed toreveal the original functional group. In some embodiments an “alcoholprotecting group” is used. An “alcohol protecting group” is any groupwhich decreases or eliminates unwanted reactivity of an alcoholfunctional group. Protecting groups can be added and removed usingtechniques well known in the art.

Synthesis of Formula A

In one embodiments, nucleic acid-lipid particles of the invention areformulated using a cationic lipid of formula A:

where R1 and R2 are independently alkyl, alkenyl or alkynyl, each can beoptionally substituted, and R3 and R4 are independently lower alkyl orR3 and R4 can be taken together to form an optionally substitutedheterocyclic ring. In some embodiments, the cationic lipid is XTC(2,2-Dilinoleyl-4-dimethylaminoethyl-[1,3]-dioxolane). In general, thelipid of formula A above may be made by the following Reaction Schemes 1or 2, wherein all substituents are as defined above unless indicatedotherwise.

Lipid A, where R₁ and R₂ are independently alkyl, alkenyl or alkynyl,each can be optionally substituted, and R₃ and R₄ are independentlylower alkyl or R₃ and R₄ can be taken together to form an optionallysubstituted heterocyclic ring, can be prepared according to Scheme 1.Ketone 1 and bromide 2 can be purchased or prepared according to methodsknown to those of ordinary skill in the art. Reaction of 1 and 2 yieldsketal 3. Treatment of ketal 3 with amine 4 yields lipids of formula A.The lipids of formula A can be converted to the corresponding ammoniumsalt with an organic salt of formula 5, where X is anion counter ionselected from halogen, hydroxide, phosphate, sulfate, or the like.

Alternatively, the ketone 1 starting material can be prepared accordingto Scheme 2. Grignard reagent 6 and cyanide 7 can be purchased orprepared according to methods known to those of ordinary skill in theart. Reaction of 6 and 7 yields ketone 1. Conversion of ketone 1 to thecorresponding lipids of formula A is as described in Scheme 1.

Synthesis of MC3

Preparation of DLin-M-C3-DMA (i.e.,(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-yl4-(dimethylamino)butanoate) was as follows. A solution of(6Z,9Z,28Z,31Z)-heptatriaconta-6,9,28,31-tetraen-19-ol (0.53 g),4-N,N-dimethylaminobutyric acid hydrochloride (0.51 g),4-N,N-dimethylaminopyridine (0.61 g) and1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (0.53 g) indichloromethane (5 mL) was stirred at room temperature overnight. Thesolution was washed with dilute hydrochloric acid followed by diluteaqueous sodium bicarbonate. The organic fractions were dried overanhydrous magnesium sulphate, filtered and the solvent removed on arotovap. The residue was passed down a silica gel column (20 g) using a1-5% methanol/dichloromethane elution gradient. Fractions containing thepurified product were combined and the solvent removed, yielding acolorless oil (0.54 g).

Synthesis of ALNY-100

Synthesis of ketal 519 [ALNY-100] was performed using the followingscheme 3:

Synthesis of 515:

To a stirred suspension of LiAlH4 (3.74 g, 0.09852 mol) in 200 mlanhydrous THF in a two neck RBF (1L), was added a solution of 514 (10 g,0.04926 mol) in 70 mL of THF slowly at 0° C. under nitrogen atmosphere.After complete addition, reaction mixture was warmed to room temperatureand then heated to reflux for 4 h. Progress of the reaction wasmonitored by TLC. After completion of reaction (by TLC) the mixture wascooled to 0° C. and quenched with careful addition of saturated Na2SO4solution. Reaction mixture was stirred for 4 h at room temperature andfiltered off. Residue was washed well with THF. The filtrate andwashings were mixed and diluted with 400 mL dioxane and 26 mL conc. HCland stirred for 20 minutes at room temperature. The volatilities werestripped off under vacuum to furnish the hydrochloride salt of 515 as awhite solid. Yield: 7.12 g ¹H-NMR (DMSO, 400 MHz): δ=9.34 (broad, 2H),5.68 (s, 2H), 3.74 (m, 1H), 2.66-2.60 (m, 2H), 2.50-2.45 (m, 5H).

Synthesis of 516:

To a stirred solution of compound 515 in 100 mL dry DCM in a 250 mL twoneck RBF, was added NEt3 (37.2 mL, 0.2669 mol) and cooled to 0° C. undernitrogen atmosphere. After a slow addition ofN-(benzyloxy-carbonyloxy)-succinimide (20 g, 0.08007 mol) in 50 mL dryDCM, reaction mixture was allowed to warm to room temperature. Aftercompletion of the reaction (2-3 h by TLC) mixture was washedsuccessively with 1N HCl solution (1×100 mL) and saturated NaHCO3solution (1×50 mL). The organic layer was then dried over anhyd. Na2SO4and the solvent was evaporated to give crude material which was purifiedby silica gel column chromatography to get 516 as sticky mass. Yield: 11g (89%). ¹H-NMR (CDCl₃, 400 MHz): δ=7.36-7.27 (m, 5H), 5.69 (s, 2H),5.12 (s, 2H), 4.96 (br., 1H) 2.74 (s, 3H), 2.60 (m, 2H), 2.30-2.25 (m,2H). LC-MS [M+H]−232.3 (96.94%).

Synthesis of 517A and 517B:

The cyclopentene 516 (5 g, 0.02164 mol) was dissolved in a solution of220 mL acetone and water (10:1) in a single neck 500 mL RBF and to itwas added N-methyl morpholine-N-oxide (7.6 g, 0.06492 mol) followed by4.2 mL of 7.6% solution of OsO4 (0.275 g, 0.00108 mol) in tert-butanolat room temperature. After completion of the reaction (˜3 h), themixture was quenched with addition of solid Na2SO3 and resulting mixturewas stirred for 1.5 h at room temperature. Reaction mixture was dilutedwith DCM (300 mL) and washed with water (2×100 mL) followed by saturatedNaHCO3 (1×50 mL) solution, water (1×30 mL) and finally with brine (1×50mL). Organic phase was dried over an.Na2SO4 and solvent was removed invacuum. Silica gel column chromatographic purification of the crudematerial was afforded a mixture of diastereomers, which were separatedby prep HPLC. Yield: −6 g crude

517A—Peak-1 (white solid), 5.13 g (96%). 1H-NMR (DMSO, 400 MHz):δ=7.39-7.31 (m, 5H), 5.04 (s, 2H), 4.78-4.73 (m, 1H), 4.48-4.47 (d, 2H),3.94-3.93 (m, 2H), 2.71 (s, 3H), 1.72-1.67 (m, 4H). LC-MS—[M+H]−266.3,[M+NH4+]−283.5 present, HPLC-97.86%. Stereochemistry confirmed by X-ray.

Synthesis of 518:

Using a procedure analogous to that described for the synthesis ofcompound 505, compound 518 (1.2 g, 41%) was obtained as a colorless oil.¹H-NMR (CDCl₃, 400 MHz): ∂=7.35-7.33 (m, 4H), 7.30-7.27 (m, 1H),5.37-5.27 (m, 8H), 5.12 (s, 2H), 4.75 (m, 1H), 4.58-4.57 (m, 2H),2.78-2.74 (m, 7H), 2.06-2.00 (m, 8H), 1.96-1.91 (m, 2H), 1.62 (m, 4H),1.48 (m, 2H), 1.37-1.25 (br m, 36H), 0.87 (m, 6H). HPLC-98.65%.

General Procedure for the Synthesis of Compound 519:

A solution of compound 518 (1 eq) in hexane (15 mL) was added in adrop-wise fashion to an ice-cold solution of LAH in THF (1 M, 2 eq).After complete addition, the mixture was heated at 40° C. over 0.5 hthen cooled again on an ice bath. The mixture was carefully hydrolyzedwith saturated aqueous Na2SO4 then filtered through celite and reducedto an oil. Column chromatography provided the pure 519 (1.3 g, 68%)which was obtained as a colorless oil. 13C NMR=130.2, 130.1 (x2), 127.9(x3), 112.3, 79.3, 64.4, 44.7, 38.3, 35.4, 31.5, 29.9 (x2), 29.7, 29.6(x2), 29.5 (x3), 29.3 (x2), 27.2 (x3), 25.6, 24.5, 23.3, 226, 14.1;Electrospray MS (+ve): Molecular weight for C44H80NO2 (M+H)+ Calc.654.6. Found 654.6.

Formulations prepared by either the standard or extrusion-free methodcan be characterized in similar manners. For example, formulations aretypically characterized by visual inspection. They should be whitishtranslucent solutions free from aggregates or sediment. Particle sizeand particle size distribution of lipid-nanoparticles can be measured bylight scattering using, for example, a Malvern Zetasizer Nano Z S(Malvern, USA). Particles should be about 20-300 nm, such as 40-100 nmin size. The particle size distribution should be unimodal. The totaldsRNA concentration in the formulation, as well as the entrappedfraction, is estimated using a dye exclusion assay. A sample of theformulated dsRNA can be incubated with an RNA-binding dye, such asRibogreen (Molecular Probes) in the presence or absence of a formulationdisrupting surfactant, e.g., 0.5% Triton-X100. The total dsRNA in theformulation can be determined by the signal from the sample containingthe surfactant, relative to a standard curve. The entrapped fraction isdetermined by subtracting the “free” dsRNA content (as measured by thesignal in the absence of surfactant) from the total dsRNA content.Percent entrapped dsRNA is typically >85%. For SNALP formulation, theparticle size is at least 30 nm, at least 40 nm, at least 50 nm, atleast 60 nm, at least 70 nm, at least 80 nm, at least 90 nm, at least100 nm, at least 110 nm, and at least 120 nm. The suitable range istypically about at least 50 nm to about at least 110 nm, about at least60 nm to about at least 100 nm, or about at least 80 nm to about atleast 90 nm.

Compositions and formulations for oral administration include powders orgranules, microparticulates, nanoparticulates, suspensions or solutionsin water or non-aqueous media, capsules, gel capsules, sachets, tabletsor minitablets. Thickeners, flavoring agents, diluents, emulsifiers,dispersing aids or binders may be desirable. In some embodiments, oralformulations are those in which dsRNAs featured in the invention areadministered in conjunction with one or more penetration enhancerssurfactants and chelators. Suitable surfactants include fatty acidsand/or esters or salts thereof, bile acids and/or salts thereof.Suitable bile acids/salts include chenodeoxycholic acid (CDCA) andursodeoxychenodeoxycholic acid (UDCA), cholic acid, dehydrocholic acid,deoxycholic acid, glucholic acid, glycholic acid, glycodeoxycholic acid,taurocholic acid, taurodeoxycholic acid, sodiumtauro-24,25-dihydro-fusidate and sodium glycodihydrofusidate. Suitablefatty acids include arachidonic acid, undecanoic acid, oleic acid,lauric acid, caprylic acid, capric acid, myristic acid, palmitic acid,stearic acid, linoleic acid, linolenic acid, dicaprate, tricaprate,monoolein, dilaurin, glyceryl 1-monocaprate,1-dodecylazacycloheptan-2-one, an acylcarnitine, an acylcholine, or amonoglyceride, a diglyceride or a pharmaceutically acceptable saltthereof (e.g., sodium). In some embodiments, combinations of penetrationenhancers are used, for example, fatty acids/salts in combination withbile acids/salts. One exemplary combination is the sodium salt of lauricacid, capric acid and UDCA. Further penetration enhancers includepolyoxyethylene-9-lauryl ether, polyoxyethylene-20-cetyl ether. DsRNAsfeatured in the invention may be delivered orally, in granular formincluding sprayed dried particles, or complexed to form micro ornanoparticles. DsRNA complexing agents include poly-amino acids;polyimines; polyacrylates; polyalkylacrylates, polyoxethanes,polyalkylcyanoacrylates; cationized gelatins, albumins, starches,acrylates, polyethyleneglycols (PEG) and starches;polyalkylcyanoacrylates; DEAE-derivatized polyimines, pollulans,celluloses and starches. Suitable complexing agents include chitosan,N-trimethylchitosan, poly-L-lysine, polyhistidine, polyornithine,polyspermines, protamine, polyvinylpyridine,polythiodiethylaminomethylethylene P(TDAE), polyaminostyrene (e.g.,p-amino), poly(methylcyanoacrylate), poly(ethylcyanoacrylate),poly(butylcyanoacrylate), poly(isobutylcyanoacrylate),poly(isohexylcynaoacrylate), DEAE-methacrylate, DEAE-hexylacrylate,DEAE-acrylamide, DEAE-albumin and DEAE-dextran, polymethylacrylate,polyhexylacrylate, poly(D,L-lactic acid), poly(DL-lactic-co-glycolicacid (PLGA), alginate, and polyethyleneglycol (PEG). Oral formulationsfor dsRNAs and their preparation are described in detail in U.S. Pat.No. 6,887,906, US Publn. No. 20030027780, and U.S. Pat. No. 6,747,014,each of which is incorporated herein by reference.

Compositions and formulations for parenteral, intraparenchymal (into thebrain), intrathecal, intraventricular or intrahepatic administration mayinclude sterile aqueous solutions which may also contain buffers,diluents and other suitable additives such as, but not limited to,penetration enhancers, carrier compounds and other pharmaceuticallyacceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids. Particularlypreferred are formulations that target the liver when treating hepaticdisorders such as hepatic carcinoma.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral, the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, gel capsules, liquid syrups, soft gels, suppositories, andenemas. The compositions of the present invention may also be formulatedas suspensions in aqueous, non-aqueous or mixed media. Aqueoussuspensions may further contain substances which increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

Additional Formulations

Emulsions

The compositions of the present invention may be prepared and formulatedas emulsions. Emulsions are typically heterogeneous systems of oneliquid dispersed in another in the form of droplets usually exceeding0.1 μm in diameter (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199; Rosoff, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245; Block inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335; Higuchi et al.,in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton,Pa., 1985, p. 301). Emulsions are often biphasic systems comprising twoimmiscible liquid phases intimately mixed and dispersed with each other.In general, emulsions may be of either the water-in-oil (w/o) or theoil-in-water (o/w) variety. When an aqueous phase is finely divided intoand dispersed as minute droplets into a bulk oily phase, the resultingcomposition is called a water-in-oil (w/o) emulsion. Alternatively, whenan oily phase is finely divided into and dispersed as minute dropletsinto a bulk aqueous phase, the resulting composition is called anoil-in-water (o/w) emulsion. Emulsions may contain additional componentsin addition to the dispersed phases, and the active drug which may bepresent as a solution in either the aqueous phase, oily phase or itselfas a separate phase. Pharmaceutical excipients such as emulsifiers,stabilizers, dyes, and anti-oxidants may also be present in emulsions asneeded. Pharmaceutical emulsions may also be multiple emulsions that arecomprised of more than two phases such as, for example, in the case ofoil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions.Such complex formulations often provide certain advantages that simplebinary emulsions do not. Multiple emulsions in which individual oildroplets of an o/w emulsion enclose small water droplets constitute aw/o/w emulsion. Likewise a system of oil droplets enclosed in globulesof water stabilized in an oily continuous phase provides an o/w/oemulsion.

Emulsions are characterized by little or no thermodynamic stability.Often, the dispersed or discontinuous phase of the emulsion is welldispersed into the external or continuous phase and maintained in thisform through the means of emulsifiers or the viscosity of theformulation. Either of the phases of the emulsion may be a semisolid ora solid, as is the case of emulsion-style ointment bases and creams.Other means of stabilizing emulsions entail the use of emulsifiers thatmay be incorporated into either phase of the emulsion. Emulsifiers maybroadly be classified into four categories: synthetic surfactants,naturally occurring emulsifiers, absorption bases, and finely dispersedsolids (see e.g., Ansel's Pharmaceutical Dosage Forms and Drug DeliverySystems, Allen, L V., Popovich N G., and Ansel H C., 2004, LippincottWilliams & Wilkins (8th ed.), New York, N.Y.; Idson, in PharmaceuticalDosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker,Inc., New York, N.Y., volume 1, p. 199).

Synthetic surfactants, also known as surface active agents, have foundwide applicability in the formulation of emulsions and have beenreviewed in the literature (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), Marcel Dekker, Inc., New York, N.Y., 1988, volume 1, p. 199).Surfactants are typically amphiphilic and comprise a hydrophilic and ahydrophobic portion. The ratio of the hydrophilic to the hydrophobicnature of the surfactant has been termed the hydrophile/lipophilebalance (HLB) and is a valuable tool in categorizing and selectingsurfactants in the preparation of formulations. Surfactants may beclassified into different classes based on the nature of the hydrophilicgroup: nonionic, anionic, cationic and amphoteric (see e.g., Ansel'sPharmaceutical Dosage Forms and Drug Delivery Systems, Allen, L V.,Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins (8thed.), New York, N.Y. Rieger, in Pharmaceutical Dosage Forms, Lieberman,Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y.,volume 1, p. 285).

Naturally occurring emulsifiers used in emulsion formulations includelanolin, beeswax, phosphatides, lecithin and acacia. Absorption basespossess hydrophilic properties such that they can soak up water to formw/o emulsions yet retain their semisolid consistencies, such asanhydrous lanolin and hydrophilic petrolatum. Finely divided solids havealso been used as good emulsifiers especially in combination withsurfactants and in viscous preparations. These include polar inorganicsolids, such as heavy metal hydroxides, nonswelling clays such asbentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidalaluminum silicate and colloidal magnesium aluminum silicate, pigmentsand nonpolar solids such as carbon or glyceryl tristearate.

A large variety of non-emulsifying materials are also included inemulsion formulations and contribute to the properties of emulsions.These include fats, oils, waxes, fatty acids, fatty alcohols, fattyesters, humectants, hydrophilic colloids, preservatives and antioxidants(Block, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 335;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).

Hydrophilic colloids or hydrocolloids include naturally occurring gumsand synthetic polymers such as polysaccharides (for example, acacia,agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth),cellulose derivatives (for example, carboxymethylcellulose andcarboxypropylcellulose), and synthetic polymers (for example, carbomers,cellulose ethers, and carboxyvinyl polymers). These disperse or swell inwater to form colloidal solutions that stabilize emulsions by formingstrong interfacial films around the dispersed-phase droplets and byincreasing the viscosity of the external phase.

Since emulsions often contain a number of ingredients such ascarbohydrates, proteins, sterols and phosphatides that may readilysupport the growth of microbes, these formulations often incorporatepreservatives. Commonly used preservatives included in emulsionformulations include methyl paraben, propyl paraben, quaternary ammoniumsalts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boricacid. Antioxidants are also commonly added to emulsion formulations toprevent deterioration of the formulation. Antioxidants used may be freeradical scavengers such as tocopherols, alkyl gallates, butylatedhydroxyanisole, butylated hydroxytoluene, or reducing agents such asascorbic acid and sodium metabisulfite, and antioxidant synergists suchas citric acid, tartaric acid, and lecithin.

The application of emulsion formulations via dermatological, oral andparenteral routes and methods for their manufacture have been reviewedin the literature (see e.g., Ansel's Pharmaceutical Dosage Forms andDrug Delivery Systems, Allen, L V., Popovich N G., and Ansel H C., 2004,Lippincott Williams & Wilkins (8th ed.), New York, N.Y.; Idson, inPharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988,Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199). Emulsionformulations for oral delivery have been very widely used because ofease of formulation, as well as efficacy from an absorption andbioavailability standpoint (see e.g., Ansel's Pharmaceutical DosageForms and Drug Delivery Systems, Allen, L V., Popovich N G., and Ansel HC., 2004, Lippincott Williams & Wilkins (8th ed.), New York, N.Y.;Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 245;Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker(Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199).Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritivepreparations are among the materials that have commonly beenadministered orally as o/w emulsions.

In one embodiment of the present invention, the compositions of iRNAsand nucleic acids are formulated as microemulsions. A microemulsion maybe defined as a system of water, oil and amphiphile which is a singleoptically isotropic and thermodynamically stable liquid solution (seee.g., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems,Allen, L V., Popovich N G., and Ansel H C., 2004, Lippincott Williams &Wilkins (8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical DosageForms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc.,New York, N.Y., volume 1, p. 245). Typically microemulsions are systemsthat are prepared by first dispersing an oil in an aqueous surfactantsolution and then adding a sufficient amount of a fourth component,generally an intermediate chain-length alcohol to form a transparentsystem. Therefore, microemulsions have also been described asthermodynamically stable, isotropically clear dispersions of twoimmiscible liquids that are stabilized by interfacial films ofsurface-active molecules (Leung and Shah, in: Controlled Release ofDrugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCHPublishers, New York, pages 185-215). Microemulsions commonly areprepared via a combination of three to five components that include oil,water, surfactant, cosurfactant and electrolyte. Whether themicroemulsion is of the water-in-oil (w/o) or an oil-in-water (o/w) typeis dependent on the properties of the oil and surfactant used and on thestructure and geometric packing of the polar heads and hydrocarbon tailsof the surfactant molecules (Schott, in Remington's PharmaceuticalSciences, Mack Publishing Co., Easton, Pa., 1985, p. 271).

The phenomenological approach utilizing phase diagrams has beenextensively studied and has yielded a comprehensive knowledge, to oneskilled in the art, of how to formulate microemulsions (see e.g.,Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, Allen, LV., Popovich N G., and Ansel H C., 2004, Lippincott Williams & Wilkins(8th ed.), New York, N.Y.; Rosoff, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 245; Block, in Pharmaceutical Dosage Forms,Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., NewYork, N.Y., volume 1, p. 335). Compared to conventional emulsions,microemulsions offer the advantage of solubilizing water-insoluble drugsin a formulation of thermodynamically stable droplets that are formedspontaneously.

Surfactants used in the preparation of microemulsions include, but arenot limited to, ionic surfactants, non-ionic surfactants, Brij 96,polyoxyethylene oleyl ethers, polyglycerol fatty acid esters,tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310),hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500),decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750),decaglycerol sequioleate (SO750), decaglycerol decaoleate (DA0750),alone or in combination with cosurfactants. The cosurfactant, usually ashort-chain alcohol such as ethanol, 1-propanol, and 1-butanol, servesto increase the interfacial fluidity by penetrating into the surfactantfilm and consequently creating a disordered film because of the voidspace generated among surfactant molecules. Microemulsions may, however,be prepared without the use of cosurfactants and alcohol-freeself-emulsifying microemulsion systems are known in the art. The aqueousphase may typically be, but is not limited to, water, an aqueoussolution of the drug, glycerol, PEG300, PEG400, polyglycerols, propyleneglycols, and derivatives of ethylene glycol. The oil phase may include,but is not limited to, materials such as Captex 300, Captex 355, CapmulMCM, fatty acid esters, medium chain (C8-C12) mono, di, andtri-glycerides, polyoxyethylated glyceryl fatty acid esters, fattyalcohols, polyglycolized glycerides, saturated polyglycolized C8-C10glycerides, vegetable oils and silicone oil.

Microemulsions are particularly of interest from the standpoint of drugsolubilization and the enhanced absorption of drugs. Lipid basedmicroemulsions (both o/w and w/o) have been proposed to enhance the oralbioavailability of drugs, including peptides (see e.g., U.S. Pat. Nos.6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides et al.,Pharmaceutical Research, 1994, 11, 1385-1390; Ritschel, Meth. Find. Exp.Clin. Pharmacol., 1993, 13, 205). Microemulsions afford advantages ofimproved drug solubilization, protection of drug from enzymatichydrolysis, possible enhancement of drug absorption due tosurfactant-induced alterations in membrane fluidity and permeability,ease of preparation, ease of oral administration over solid dosageforms, improved clinical potency, and decreased toxicity (see e.g., U.S.Pat. No. 6,191,105; 7,063,860; 7,070,802; 7,157,099; Constantinides etal., Pharmaceutical Research, 1994, 11, 1385; Ho et al., J. Pharm. Sci.,1996, 85, 138-143). Often microemulsions may form spontaneously whentheir components are brought together at ambient temperature. This maybe particularly advantageous when formulating thermolabile drugs,peptides or iRNAs. Microemulsions have also been effective in thetransdermal delivery of active components in both cosmetic andpharmaceutical applications. It is expected that the microemulsioncompositions and formulations of the present invention will facilitatethe increased systemic absorption of iRNAs and nucleic acids from thegastrointestinal tract, as well as improve the local cellular uptake ofiRNAs and nucleic acids.

Microemulsions of the present invention may also contain additionalcomponents and additives such as sorbitan monostearate (Grill 3),Labrasol, and penetration enhancers to improve the properties of theformulation and to enhance the absorption of the iRNAs and nucleic acidsof the present invention. Penetration enhancers used in themicroemulsions of the present invention may be classified as belongingto one of five broad categories—surfactants, fatty acids, bile salts,chelating agents, and non-chelating non-surfactants (Lee et al.,Critical Reviews in Therapeutic Drug Carrier Systems, 1991, p. 92). Eachof these classes has been discussed above.

Penetration Enhancers

In one embodiment, the present invention employs various penetrationenhancers to effect the efficient delivery of nucleic acids,particularly iRNAs, to the skin of animals. Most drugs are present insolution in both ionized and nonionized forms. However, usually onlylipid soluble or lipophilic drugs readily cross cell membranes. It hasbeen discovered that even non-lipophilic drugs may cross cell membranesif the membrane to be crossed is treated with a penetration enhancer. Inaddition to aiding the diffusion of non-lipophilic drugs across cellmembranes, penetration enhancers also enhance the permeability oflipophilic drugs.

Penetration enhancers may be classified as belonging to one of fivebroad categories, i.e., surfactants, fatty acids, bile salts, chelatingagents, and non-chelating non-surfactants (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92). Each of the above mentioned classes ofpenetration enhancers are described below in greater detail.

Surfactants:

In connection with the present invention, surfactants (or“surface-active agents”) are chemical entities which, when dissolved inan aqueous solution, reduce the surface tension of the solution or theinterfacial tension between the aqueous solution and another liquid,with the result that absorption of iRNAs through the mucosa is enhanced.In addition to bile salts and fatty acids, these penetration enhancersinclude, for example, sodium lauryl sulfate, polyoxyethylene-9-laurylether and polyoxyethylene-20-cetyl ether) (see e.g., Malmsten, M.Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Lee et al., Critical Reviews in Therapeutic DrugCarrier Systems, 1991, p. 92); and perfluorochemical emulsions, such asFC-43. Takahashi et al., J. Pharm. Pharmacol., 1988, 40, 252).

Fatty Acids:

Various fatty acids and their derivatives which act as penetrationenhancers include, for example, oleic acid, lauric acid, capric acid(n-decanoic acid), myristic acid, palmitic acid, stearic acid, linoleicacid, linolenic acid, dicaprate, tricaprate, monoolein(1-monooleoyl-rac-glycerol), dilaurin, caprylic acid, arachidonic acid,glycerol 1-monocaprate, 1-dodecylazacycloheptan-2-one, acylcarnitines,acylcholines, C₁₋₂₀ alkyl esters thereof (e.g., methyl, isopropyl andt-butyl), and mono- and di-glycerides thereof (i.e., oleate, laurate,caprate, myristate, palmitate, stearate, linoleate, etc.) (see e.g.,Touitou, E., et al. Enhancement in Drug Delivery, CRC Press, Danvers,Mass., 2006; Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, p. 92; Muranishi, Critical Reviews in Therapeutic DrugCarrier Systems, 1990, 7, 1-33; El Hariri et al., J. Pharm. Pharmacol.,1992, 44, 651-654).

Bile Salts:

The physiological role of bile includes the facilitation of dispersionand absorption of lipids and fat-soluble vitamins (see e.g., Malmsten,M. Surfactants and polymers in drug delivery, Informa Health Care, NewYork, N.Y., 2002; Brunton, Chapter 38 in: Goodman & Gilman's ThePharmacological Basis of Therapeutics, 9th Ed., Hardman et al. Eds.,McGraw-Hill, New York, 1996, pp. 934-935). Various natural bile salts,and their synthetic derivatives, act as penetration enhancers. Thus theterm “bile salts” includes any of the naturally occurring components ofbile as well as any of their synthetic derivatives. Suitable bile saltsinclude, for example, cholic acid (or its pharmaceutically acceptablesodium salt, sodium cholate), dehydrocholic acid (sodiumdehydrocholate), deoxycholic acid (sodium deoxycholate), glucholic acid(sodium glucholate), glycholic acid (sodium glycocholate),glycodeoxycholic acid (sodium glycodeoxycholate), taurocholic acid(sodium taurocholate), taurodeoxycholic acid (sodium taurodeoxycholate),chenodeoxycholic acid (sodium chenodeoxycholate), ursodeoxycholic acid(UDCA), sodium tauro-24,25-dihydro-fusidate (STDHF), sodiumglycodihydrofusidate and polyoxyethylene-9-lauryl ether (POE) (see e.g.,Malmsten, M. Surfactants and polymers in drug delivery, Informa HealthCare, New York, N.Y., 2002; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Swinyard, Chapter 39 In:Remington's Pharmaceutical Sciences, 18th Ed., Gennaro, ed., MackPublishing Co., Easton, Pa., 1990, pages 782-783; Muranishi, CriticalReviews in Therapeutic Drug Carrier Systems, 1990, 7, 1-33; Yamamoto etal., J. Pharm. Exp. Ther., 1992, 263, 25; Yamashita et al., J. Pharm.Sci., 1990, 79, 579-583).

Chelating Agents:

Chelating agents, as used in connection with the present invention, canbe defined as compounds that remove metallic ions from solution byforming complexes therewith, with the result that absorption of iRNAsthrough the mucosa is enhanced. With regards to their use as penetrationenhancers in the present invention, chelating agents have the addedadvantage of also serving as DNase inhibitors, as most characterized DNAnucleases require a divalent metal ion for catalysis and are thusinhibited by chelating agents (Jarrett, J. Chromatogr., 1993, 618,315-339). Suitable chelating agents include but are not limited todisodium ethylenediaminetetraacetate (EDTA), citric acid, salicylates(e.g., sodium salicylate, 5-methoxysalicylate and homovanilate), N-acylderivatives of collagen, laureth-9 and N-amino acyl derivatives ofbeta-diketones (enamines)(see e.g., Katdare, A. et al., Excipientdevelopment for pharmaceutical, biotechnology, and drug delivery, CRCPress, Danvers, Mass., 2006; Lee et al., Critical Reviews in TherapeuticDrug Carrier Systems, 1991, page 92; Muranishi, Critical Reviews inTherapeutic Drug Carrier Systems, 1990, 7, 1-33; Buur et al., J. ControlRel., 1990, 14, 43-51).

Non-Chelating Non-Surfactants:

As used herein, non-chelating non-surfactant penetration enhancingcompounds can be defined as compounds that demonstrate insignificantactivity as chelating agents or as surfactants but that nonethelessenhance absorption of iRNAs through the alimentary mucosa (see e.g.,Muranishi, Critical Reviews in Therapeutic Drug Carrier Systems, 1990,7, 1-33). This class of penetration enhancers include, for example,unsaturated cyclic ureas, 1-alkyl- and 1-alkenylazacyclo-alkanonederivatives (Lee et al., Critical Reviews in Therapeutic Drug CarrierSystems, 1991, page 92); and non-steroidal anti-inflammatory agents suchas diclofenac sodium, indomethacin and phenylbutazone (Yamashita et al.,J. Pharm. Pharmacol., 1987, 39, 621-626).

Agents that enhance uptake of iRNAs at the cellular level may also beadded to the pharmaceutical and other compositions of the presentinvention. For example, cationic lipids, such as lipofectin (Junichi etal, U.S. Pat. No. 5,705,188), cationic glycerol derivatives, andpolycationic molecules, such as polylysine (Lollo et al., PCTApplication WO 97/30731), are also known to enhance the cellular uptakeof dsRNAs. Examples of commercially available transfection reagentsinclude, for example Lipofectamine™ (Invitrogen; Carlsbad, Calif.),Lipofectamine 2000™ (Invitrogen; Carlsbad, Calif.), 293fectin™(Invitrogen; Carlsbad, Calif.), Cellfectin™ (Invitrogen; Carlsbad,Calif.), DMRIE-C™ (Invitrogen; Carlsbad, Calif.), FreeStyle™ MAX(Invitrogen; Carlsbad, Calif.), Lipofectamine™ 2000 CD (Invitrogen;Carlsbad, Calif.), Lipofectamine™ (Invitrogen; Carlsbad, Calif.),RNAiMAX (Invitrogen; Carlsbad, Calif.), Oligofectamine™ (Invitrogen;Carlsbad, Calif.), Optifect™ (Invitrogen; Carlsbad, Calif.), X-tremeGENEQ2 Transfection Reagent (Roche; Grenzacherstrasse, Switzerland), DOTAPLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), DOSPERLiposomal Transfection Reagent (Grenzacherstrasse, Switzerland), orFugene (Grenzacherstrasse, Switzerland), Transfectam® Reagent (Promega;Madison, Wis.), TransFast™ Transfection Reagent (Promega; Madison,Wis.), Tfx™-20 Reagent (Promega; Madison, Wis.), Tfx™-50 Reagent(Promega; Madison, Wis.), DreamFect™ (OZ Biosciences; Marseille,France), EcoTransfect (OZ Biosciences; Marseille, France), TransPass^(a)D1 Transfection Reagent (New England Biolabs; Ipswich, Mass., USA),LyoVec™/LipoGen™ (Invivogen; San Diego, Calif., USA), PerFectinTransfection Reagent (Genlantis; San Diego, Calif., USA), NeuroPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), GenePORTERTransfection reagent (Genlantis; San Diego, Calif., USA), GenePORTER 2Transfection reagent (Genlantis; San Diego, Calif., USA), CytofectinTransfection Reagent (Genlantis; San Diego, Calif., USA), BaculoPORTERTransfection Reagent (Genlantis; San Diego, Calif., USA), TroganPORTERT™transfection Reagent (Genlantis; San Diego, Calif., USA), RiboFect(Bioline; Taunton, Mass., USA), PlasFect (Bioline; Taunton, Mass., USA),UniFECTOR (B-Bridge International; Mountain View, Calif., USA),SureFECTOR (B-Bridge International; Mountain View, Calif., USA), orHiFect™ (B-Bridge International, Mountain View, Calif., USA), amongothers.

Other agents may be utilized to enhance the penetration of theadministered nucleic acids, including glycols such as ethylene glycoland propylene glycol, pyrrols such as 2-pyrrol, azones, and terpenessuch as limonene and menthone.

Carriers

Certain compositions of the present invention also incorporate carriercompounds in the formulation. As used herein, “carrier compound” or“carrier” can refer to a nucleic acid, or analog thereof, which is inert(i.e., does not possess biological activity per se) but is recognized asa nucleic acid by in vivo processes that reduce the bioavailability of anucleic acid having biological activity by, for example, degrading thebiologically active nucleic acid or promoting its removal fromcirculation. The coadministration of a nucleic acid and a carriercompound, typically with an excess of the latter substance, can resultin a substantial reduction of the amount of nucleic acid recovered inthe liver, kidney or other extracirculatory reservoirs, presumably dueto competition between the carrier compound and the nucleic acid for acommon receptor. For example, the recovery of a partiallyphosphorothioate dsRNA in hepatic tissue can be reduced when it iscoadministered with polyinosinic acid, dextran sulfate, polycytidic acidor 4-acetamido-4′isothiocyano-stilbene-2,2′-disulfonic acid (Miyao etal., DsRNA Res. Dev., 1995, 5, 115-121; Takakura et al., DsRNA & Nucl.Acid Drug Dev., 1996, 6, 177-183.

Excipients

In contrast to a carrier compound, a “pharmaceutical carrier” or“excipient” is a pharmaceutically acceptable solvent, suspending agentor any other pharmacologically inert vehicle for delivering one or morenucleic acids to an animal. The excipient may be liquid or solid and isselected, with the planned manner of administration in mind, so as toprovide for the desired bulk, consistency, etc., when combined with anucleic acid and the other components of a given pharmaceuticalcomposition. Typical pharmaceutical carriers include, but are notlimited to, binding agents (e.g., pregelatinized maize starch,polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers(e.g., lactose and other sugars, microcrystalline cellulose, pectin,gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calciumhydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc,silica, colloidal silicon dioxide, stearic acid, metallic stearates,hydrogenated vegetable oils, corn starch, polyethylene glycols, sodiumbenzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodiumstarch glycolate, etc.); and wetting agents (e.g., sodium laurylsulphate, etc).

Pharmaceutically acceptable organic or inorganic excipients suitable fornon-parenteral administration which do not deleteriously react withnucleic acids can also be used to formulate the compositions of thepresent invention. Suitable pharmaceutically acceptable carriersinclude, but are not limited to, water, salt solutions, alcohols,polyethylene glycols, gelatin, lactose, amylose, magnesium stearate,talc, silicic acid, viscous paraffin, hydroxymethylcellulose,polyvinylpyrrolidone and the like.

Formulations for topical administration of nucleic acids may includesterile and non-sterile aqueous solutions, non-aqueous solutions incommon solvents such as alcohols, or solutions of the nucleic acids inliquid or solid oil bases. The solutions may also contain buffers,diluents and other suitable additives. Pharmaceutically acceptableorganic or inorganic excipients suitable for non-parenteraladministration which do not deleteriously react with nucleic acids canbe used.

Suitable pharmaceutically acceptable excipients include, but are notlimited to, water, salt solutions, alcohol, polyethylene glycols,gelatin, lactose, amylose, magnesium stearate, talc, silicic acid,viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and thelike.

Other Components

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions,at their art-established usage levels. Thus, for example, thecompositions may contain additional, compatible, pharmaceutically-activematerials such as, for example, antipruritics, astringents, localanesthetics or anti-inflammatory agents, or may contain additionalmaterials useful in physically formulating various dosage forms of thecompositions of the present invention, such as dyes, flavoring agents,preservatives, antioxidants, opacifiers, thickening agents andstabilizers. However, such materials, when added, should not undulyinterfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the nucleic acid(s) of the formulation.

Aqueous suspensions may contain substances that increase the viscosityof the suspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In some embodiments, pharmaceutical compositions featured in theinvention include (a) one or more iRNA compounds and (b) one or morebiologic agents which function by a non-RNAi mechanism.

Toxicity and therapeutic efficacy of such compounds can be determined bystandard pharmaceutical procedures in cell cultures or experimentalanimals, e.g., for determining the LD50 (the dose lethal to 50% of thepopulation) and the ED50 (the dose therapeutically effective in 50% ofthe population). The dose ratio between toxic and therapeutic effects isthe therapeutic index and it can be expressed as the ratio LD50/ED50.Compounds that exhibit high therapeutic indices are preferred.

The data obtained from cell culture assays and animal studies can beused in formulating a range of dosage for use in humans. The dosage ofcompositions featured in the invention lies generally within a range ofcirculating concentrations that include the ED50 with little or notoxicity. The dosage may vary within this range depending upon thedosage form employed and the route of administration utilized. For anycompound used in the methods featured in the invention, thetherapeutically effective dose can be estimated initially from cellculture assays. A dose may be formulated in animal models to achieve acirculating plasma concentration range of the compound or, whenappropriate, of the polypeptide product of a target sequence (e.g.,achieving a decreased concentration of the polypeptide) that includesthe IC50 (i.e., the concentration of the test compound which achieves ahalf-maximal inhibition of symptoms) as determined in cell culture. Suchinformation can be used to more accurately determine useful doses inhumans. Levels in plasma may be measured, for example, by highperformance liquid chromatography.

In addition to their administration, as discussed above, the iRNAsfeatured in the invention can be administered in combination with otherknown agents effective in treatment of pathological processes mediatedby EGLN expression. In any event, the administering physician can adjustthe amount and timing of iRNA administration on the basis of resultsobserved using standard measures of efficacy known in the art ordescribed herein.

Methods for Treating Diseases Caused by Expression of an EGLN Gene

The invention relates in particular to the use of an iRNA targeting EGLNand compositions containing at least one such iRNA for the treatment ofan EGLN—mediated disorder or disease. For example, a compositioncontaining an iRNA targeting at least one EGLN gene is used fortreatment of anemia. As used herein, “anemia” refers to a conditionwhereby the body has fewer than necessary red blood cells therebyresulting in reduced oxygen to cells and tissues. Anemias may be causedby any of several disorders and include, but are not limited to anemiadue to B12 deficiency, anemia due to folate deficiency, anemia due toiron deficiency, hemolytic anemia, hemolytic anemia due to G-6-PDdeficiency, idiopathic aplastic anemia, idiopathic autoimmune hemolyticanemia, immune hemolytic anemia, iegaloblastic anemia, perniciousanemia, secondary aplastic anemia, and sickle cell anemia. Certainsymptoms are associated with anemia and include pale skin, dizziness,fatigue, headaches, irritability, low body temperature, numb/cold handsor feet, rapid heartbeat, shortness of breath, weakness and chest painany of which may be ameliorated by administration of the iRNA agentstargeting one or more EGLN genes of the present invention.

In one embodiment at least one iRNA targeting at least one EGLN gene isused to downregulate hepcidin (GenBank Reference NG_(—)011563.1; SEQ ID2805 representing the complete gene on chromosome 19; and GenBankReference NM_(—)021175 representing the Hepcidin peptide; SEQ ID NO:2806). Probes for the detection of hepcidin (HAMP1) were purchased fromPanomics (a division of Affymetrix, Santa Clara, Calif.) and can detecteither HAMP1 or HAMP2. Hepcidin is a peptide hormone that is produced bythe liver. It is believed that hepcidin binds to ion channel to inhibitiron transport out of the cells which store iron. The downregulation ofhepcidin may result in increased mobilization of iron in the body.

In one embodiment at least one iRNA targeting at least one EGLN gene isused for the treatment of cancer. As used herein “cancer” refers to anyof various malignant neoplasms characterized by the proliferation ofanaplastic cells that tend to invade surrounding tissue and metastasizeto new body sites and also refers to the pathological conditioncharacterized by such malignant neoplastic growths. A cancer can be atumor or hematological malignancy, and includes but is not limited to,all types of cancers but preferably leukemias, and those arising in theblood or bone.

Leukemias, or cancers of the blood or bone marrow that are characterizedby an abnormal proliferation of white blood cells i.e., leukocytes, canbe divided into four major classifications including Acute lymphoblasticleukemia (ALL), Chronic lymphocytic leukemia (CLL), Acute myelogenousleukemia or acute myeloid leukemia (AML) (AML with translocationsbetween chromosome 10 and 11 [t(10, 11)], chromosome 8 and 21 [t(8;21)],chromosome 15 and 17 [t(15;17)], and inversions in chromosome 16[inv(16)]; AML with multilineage dysplasia, which includes patients whohave had a prior myelodysplastic syndrome (MDS) or myeloproliferativedisease that transforms into AML; AML and myelodysplastic syndrome(MDS), therapy-related, which category includes patients who have hadprior chemotherapy and/or radiation and subsequently develop AML or MDS;d) AML not otherwise categorized, which includes subtypes of AML that donot fall into the above categories; and e) Acute leukemias of ambiguouslineage, which occur when the leukemic cells can not be classified aseither myeloid or lymphoid cells, or where both types of cells arepresent); and Chronic myelogenous leukemia (CML). These types ofleukemias are particularly amenable to treatment with the iRNA agents ofthe present invention.

The invention further relates to the use of an iRNA or a pharmaceuticalcomposition thereof, e.g., for treating anemia or cancer, in combinationwith other pharmaceuticals and/or other therapeutic methods, e.g., withknown pharmaceuticals and/or known therapeutic methods, such as, forexample, those which are currently employed for treating thesedisorders. For example, the iRNA or pharmaceutical composition thereofcan also be administered in conjunction with one or more additionalanti-cancer treatments, such as biological, chemotherapy andradiotherapy. Accordingly, a treatment can include, for example,imatinib (Gleevac), all-trans-retinoic acid, a monoclonal antibodytreatment (gemtuzumab, ozogamicin), chemotherapy (for example,chlorambucil, prednisone, prednisolone, vincristine, cytarabine,clofarabine, farnesyl transferase inhibitors, decitabine, inhibitors ofMDR1), rituximab, interferon-α, anthracycline drugs (such asdaunorubicin or idarubicin), L-asparaginase, doxorubicin,cyclophosphamide, doxorubicin, bleomycin, fludarabine, etoposide,pentostatin, or cladribine), bone marrow transplant, stem celltransplant, radiation thereapy, anti-metabolite drugs (methotrexate and6-mercaptopurine), or any combination thereof.

In one embodiment, the iRNA agents of the present invention may beadministered in combination with an iron supplement. The administrationmay be simultaneously, together, or apart. The dosing may be on the sameschedule, an offset schedule or a one time administration of the ironsupplement. The iron supplement may be given on an “as needed” basisdepending on measurements made in the particular patient.

Radiation therapy (also called radiotherapy, X-ray therapy, orirradiation) is the use of ionizing radiation to kill cancer cells andshrink tumors. Radiation therapy can be administered externally viaexternal beam radiotherapy (EBRT) or internally via brachytherapy. Theeffects of radiation therapy are localised and confined to the regionbeing treated. Radiation therapy may be used to treat almost every typeof solid tumor, including cancers of the brain, breast, cervix, larynx,lung, pancreas, prostate, skin, stomach, uterus, or soft tissuesarcomas. Radiation is also used to treat leukemia and lymphoma.

Chemotherapy is the treatment of cancer with drugs that can destroycancer cells. In current usage, the term “chemotherapy” usually refersto cytotoxic drugs which affect rapidly dividing cells in general, incontrast with targeted therapy. Chemotherapy drugs interfere with celldivision in various possible ways, e.g. with the duplication of DNA orthe separation of newly formed chromosomes. Most forms of chemotherapytarget all rapidly dividing cells and are not specific to cancer cells,although some degree of specificity may come from the inability of manycancer cells to repair DNA damage, while normal cells generally can.Most chemotherapy regimens are given in combination. Exemplarychemotherapeutic agents include, but are not limited to, 5-FU Enhancer,9-AC, AG2037, AG3340, Aggrecanase Inhibitor, Aminoglutethimide,Amsacrine (m-AMSA), Asparaginase, Azacitidine, Batimastat (BB94), BAY12-9566, BCH-4556, Bis-Naphtalimide, Busulfan, Capecitabine,Carboplatin, Carmustaine+Polifepr Osan, cdk4/cdk2 inhibitors,Chlorombucil, CI-994, Cisplatin, Cladribine, CS-682, Cytarabine HCl,D2163, Dactinomycin, Daunorubicin HCl, DepoCyt, Dexifosamide, Docetaxel,Dolastain, Doxifluridine, Doxorubicin, DX8951f, E 7070, EGFR,Epirubicin, Erythropoietin, Estramustine phosphate sodium, Etoposide(VP16-213), Farnesyl Transferase Inhibitor, FK 317, Flavopiridol,Floxuridine, Fludarabine, Fluorouracil (5-FU), Flutamide, Fragyline,Gemcitabine, Hexamethylmelamine (HMM), Hydroxyurea (hydroxycarbamide),Ifosfamide, Interferon Alfa-2a, Interferon Alfa-2b, Interleukin-2,Irinotecan, ISI 641, Krestin, Lemonal DP 2202, Leuprolide acetate(LHRH-releasing factor analogue), Levamisole, LiGLA (lithium-gammalinolenate), Lodine Seeds, Lometexol, Lomustine (CCNU), Marimistat,Mechlorethamine HCl (nitrogen mustard), Megestrol acetate, MeglamineGLA, Mercaptopurine, Mesna, Mitoguazone (methyl-GAG; methyl glyoxalbis-guanylhydrazone; MGBG), Mitotane (o.p′-DDD), Mitoxantrone,Mitoxantrone HCl, MMI 270, MMP, MTA/LY 231514, Octreotide, ODN 698,OK-432, Oral Platinum, Oral Taxoid, Paclitaxel (TAXOL®), PARPInhibitors, PD 183805, Pentostatin (2′ deoxycoformycin), PKC 412,Plicamycin, Procarbazine HCl, PSC 833, Ralitrexed, RAS FarnesylTransferase Inhibitor, RAS Oncogene Inhibitor, Semustine (methyl-CCNU),Streptozocin, Suramin, Tamoxifen citrate, Taxane Analog, Temozolomide,Teniposide (VM-26), Thioguanine, Thiotepa, Topotecan, Tyrosine Kinase,UFT (Tegafur/Uracil), Valrubicin, Vinblastine sulfate, Vindesinesulfate, VX-710, VX-853, YM 116, ZD 0101, ZD 0473/Anormed, ZD 1839, ZD9331.

Biological therapies use the body's immune system, either directly orindirectly, to fight cancer or to lessen the side effects that may becaused by some cancer treatments. In one sense, targeting one or moreEGLN genes can be considered in this group of therapies in that it canstimulate immune system action against a tumor, for example. However,this approach can also be considered with other such biologicalapproaches, e.g., immune response modifying therapies such as theadministration of interferons, interleukins, colony-stimulating factors,monoclonal antibodies, vaccines, gene therapy, and nonspecificimmunomodulating agents are also envisioned as anti-cancer therapies tobe combined with the inhibition of EGLN. Small molecule targeted therapydrugs are generally inhibitors of enzymatic domains on mutated,overexpressed, or otherwise critical proteins within the cancer cell,such as tyrosine kinase inhibitors imatinib (Gleevec/Glivec) andgefitinib (Iressa). Examples of monoclonal antibody therapies that canbe used with an iRNA or pharmaceutical composition thereof include, butare not limited to, the anti-HER2/neu antibody trastuzumab (Herceptin)used in breast cancer, and the anti-CD20 antibody rituximab, used in avariety of B-cell malignancies. The growth of some cancers can beinhibited by providing or blocking certain hormones. Common examples ofhormone-sensitive tumors include certain types of breast and prostatecancers. Removing or blocking estrogen or testosterone is often animportant additional treatment. In certain cancers, administration ofhormone agonists, such as progestogens may be therapeuticallybeneficial.

Cancer immunotherapy refers to a diverse set of therapeutic strategiesdesigned to induce the patient's own immune system to fight the tumor,and include, but are not limited to, intravesical BCG immunotherapy forsuperficial bladder cancer, vaccines to generate specific immuneresponses, such as for malignant melanoma and renal cell carcinoma, andthe use of Sipuleucel-T for prostate cancer, in which dendritic cellsfrom the patient are loaded with prostatic acid phosphatase peptides toinduce a specific immune response against prostate-derived cells.

In some embodiments, an iRNA targeting one or more EGLN genes isadministered in combination with an angiogenesis inhibitor. In someembodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include, but are not limited to, monoclonal antibodytherapies directed against specific pro-angiogenic growth factors and/ortheir receptors. Examples of these are: bevacizumab (Avastin®),cetuximab (Erbitux®), panitumumab (Vectibix™), and trastuzumab(Herceptin®). In some embodiments, the angiogenesis inhibitors for usein the methods described herein include but are not limited to smallmolecule tyrosine kinase inhibitors (TKIs) of multiple pro-angiogenicgrowth factor receptors. The three TKIs that are currently approved asanti-cancer therapies are erlotinib (Tarceva®), sorafenib (Nexavar®),and sunitinib (Sutent®). In some embodiments, the angiogenesisinhibitors for use in the methods described herein include but are notlimited to inhibitors of mTOR (mammalian target of rapamycin) such astemsirolimus (Toricel™), bortezomib (Velcade®), thalidomide (Thalomid®),and Doxycyclin.

In other embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include one or more drugs that target the VEGF pathway,including, but not limited to, Bevacizumab (Avastin®), sunitinib(Sutent®), and sorafenib (Nexavar®). Additional VEGF inhibitors includeCP-547,632 (3-(4-Bromo-2,6-difluoro-benzyloxy)-5-[3-(4-pyrrolidin1-yl-butyl)-ureido]-isothiazole-4-carboxylic acid amide hydrochloride;Pfizer Inc., NY), AG13736, AG28262 (Pfizer Inc.), SU5416, SU11248, &SU6668 (formerly Sugen Inc., now Pfizer, New York, N.Y.), ZD-6474(AstraZeneca), ZD4190 which inhibits VEGF-R2 and -R1 (AstraZeneca),CEP-7055 (Cephalon Inc., Frazer, Pa.), PKC 412 (Novartis), AEE788(Novartis), AZD-2171), NEXAVAR® (BAY 43-9006, sorafenib; BayerPharmaceuticals and Onyx Pharmaceuticals), vatalanib (also known asPTK-787, ZK-222584: Novartis & Schering: AG), MACUGEN® (pegaptaniboctasodium, NX-1838, EYE-001, Pfizer Inc./Gilead/Eyetech), IM862(glufanide disodium, Cytran Inc. of Kirkland, Wash., USA),VEGFR2-selective monoclonal antibody DC101 (ImClone Systems, Inc.),angiozyme, a synthetic ribozyme from Ribozyme (Boulder, Colo.) andChiron (Emeryville, California), Sirna-027 (an siRNA-based VEGFR1inhibitor, Sirna Therapeutics, San Francisco, Calif.) Caplostatin,soluble ectodomains of the VEGF receptors, Neovastat (1Eterna ZentarisInc; Quebec City, Calif.), ZM323881 (CalBiochem. CA, USA), pegaptanib(Macugen) (Eyetech Pharmaceuticals), an anti-VEGF aptamer andcombinations thereof.

In other embodiments, the angiogenesis inhibitors for use in the methodsdescribed herein include anti-angiogenic factors such as alpha-2antiplasmin (fragment), angiostatin (plasminogen fragment),antiangiogenic antithrombin III, cartilage-derived inhibitor (CDI), CD59complement fragment, endostatin (collagen XVIII fragment), fibronectinfragment, gro-beta (a C—X—C chemokine), heparinases heparinhexasaccharide fragment, human chorionic gonadotropin (hCG), interferonalpha/beta/gamma, interferon inducible protein (IP-10), interleukin-12,kringle 5 (plasminogen fragment), beta-thromboglobulin, EGF (fragment),VEGF inhibitor, endostatin, fibronection (45 kD fragment), highmolecular weight kininogen (domain 5), NK1, NK2, NK3 fragments of HGF,PF-4, serpin proteinase inhibitor 8, TGF-beta-1, thrombospondin-1,prosaposin, p53, angioarrestin, metalloproteinase inhibitors (TIMPs),2-Methoxyestradiol, placental ribonuclease inhibitor, plasminogenactivator inhibitor, prolactin 16 kD fragment, proliferin-relatedprotein (PRP), retinoids, tetrahydrocortisol-S transforming growthfactor-beta (TGF-b), vasculostatin, and vasostatin (calreticulinfragment).pamidronate thalidomide, TNP470, the bisphosphonate familysuch as amino-bisphosphonate zoledronic acid. bombesin/gastrin-releasingpeptide (GRP) antagonists such as RC-3095 and RC-3940-II (Bajol A M, et.al., British Journal of Cancer (2004) 90, 245-252), anti-VEGF peptide(dRK6) (Seung-Ah Yoo, J. Immuno, 2005, 174: 5846-5855).

Efficacy of treatment or amelioration of disease can be assessed, forexample by measuring disease progression, disease remission, symptomseverity, reduction in pain, quality of life, dose of a medicationrequired to sustain a treatment effect, level of a disease marker or anyother measurable parameter appropriate for a given disease being treatedor targeted for prevention. It is well within the ability of one skilledin the art to monitor efficacy of treatment or prevention by measuringany one of such parameters, or any combination of parameters. Inconnection with the administration of an iRNA targeting one or more EGLNgenes or pharmaceutical composition thereof, “effective against” acancer indicates that administration in a clinically appropriate mannerresults in a beneficial effect for at least a statistically significantfraction of patients, such as a improvement of symptoms, a cure, areduction in disease load, reduction in tumor mass or cell numbers,extension of life, improvement in quality of life, or other effectgenerally recognized as positive by medical doctors familiar withtreating the particular type of cancer.

In one embodiment the disorder is anemia where efficacy of treatment canbe determined by measuring standard endpoints associated withimprovement anemia due to B12 deficiency, anemia due to folatedeficiency, anemia due to iron deficiency, hemolytic anemia, hemolyticanemia due to G-6-PD deficiency, idiopathic aplastic anemia, idiopathicautoimmune hemolytic anemia, immune hemolytic anemia, iegaloblasticanemia, pernicious anemia, secondary aplastic anemia, and sickle cellanemia. For example, an improvement in any of the manifestations ofanemia such as pale skin, dizziness, fatigue, headaches, irritability,low body temperature, numb/cold hands or feet, rapid heartbeat, reducederythropoietin, shortness of breath, weakness and chest pain would beconsidered indicative of effective treatment.

A treatment or preventive effect is evident when there is astatistically significant improvement in one or more parameters ofdisease status, or by a failure to worsen or to develop symptoms wherethey would otherwise be anticipated. As an example, a favorable changeof at least 10% in a measurable parameter of disease, and preferably atleast 20%, 30%, 40%, 50% or more can be indicative of effectivetreatment. Efficacy for a given iRNA drug or formulation of that drugcan also be judged using an experimental animal model for the givendisease as known in the art. When using an experimental animal model,efficacy of treatment is evidenced when a statistically significantreduction in a marker or symptom is observed.

The invention relates in particular to the use of one or more iRNAtargeting one or more EGLN genes and compositions containing at leastone such iRNA for the treatment of an EGLN-mediated disorder or disease.For example, a composition containing an iRNA targeting an EGLN gene isused for treatment of an infectious disease or disorder, for example, ina subject having an infection. In some preferred embodiments the subjecthas an infection or is at risk of having an infection. An “infection” asused herein refers to a disease or condition attributable to thepresence in a host of a foreign organism or agent that reproduces withinthe host. Infections typically involve breach of a normal mucosal orother tissue barrier by an infectious organism or agent. A subject thathas an infection is a subject having objectively measurable infectiousorganisms or agents present in the subject's body. A subject at risk ofhaving an infection is a subject that is predisposed to develop aninfection. Such a subject can include, for example, a subject with aknown or suspected exposure to an infectious organism or agent. Asubject at risk of having an infection also can include a subject with acondition associated with impaired ability to mount an immune responseto an infectious organism or agent, e.g., a subject with a congenital oracquired immunodeficiency, a subject undergoing radiation therapy orchemotherapy, a subject with a burn injury, a subject with a traumaticinjury, a subject undergoing surgery or other invasive medical or dentalprocedure.

Infections are broadly classified as bacterial, viral, fungal, orparasitic based on the category of infectious organism or agentinvolved. Other less common types of infection are also known in theart, including, e.g., infections involving rickettsiae, mycoplasmas, andagents causing scrapie, bovine spongiform encephalopthy (BSE), and priondiseases (e.g., kuru and Creutzfeldt-Jacob disease). Examples ofbacteria, viruses, fungi, and parasites which cause infection are wellknown in the art. An infection can be acute, subacute, chronic, orlatent, and it can be localized or systemic. As defined herein, a“chronic infection” refers to those infections that are not cleared bythe normal actions of the innate or adaptive immune responses andpersist in the subject for a long duration of time, on the order ofweeks, months, and years. A chronic infection may reflect latency of theinfectious agent, and may be include periods in which no infectioussymptoms are present, i.e., asymptomatic periods. Examples of chronicinfections include, but are not limited to, HIV infection andherpesvirus infections. Furthermore, an infection can be predominantlyintracellular or extracellular during at least one phase of theinfectious organism's or agent's life cycle in the host.

Exemplary viruses include, but are not limited to: Retroviridae (e.g.,human immunodeficiency viruses, such as HIV-1 (also referred to asHTLV-III), HIV-2, LAV or HTLV-III/LAV, or HIV-III, and other isolates,such as HIV-LP; Picornaviridae (e.g., polio viruses, hepatitis A virus;enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses);Calciviridae (e.g., strains that cause gastroenteritis); Togaviridae(e.g., equine encephalitis viruses, rubella viruses); Flaviviridae(e.g., dengue viruses, encephalitis viruses, yellow fever viruses);Coronaviridae (e.g., coronaviruses); Rhabdoviridae (e.g., vesicularstomatitis viruses, rabies viruses); Filoviridae (e.g., ebola viruses);Paramyxoviridae (e.g., parainfluenza viruses, mumps virus, measlesvirus, respiratory syncytial virus); adenovirus; Orthomyxoviridae (e.g.,influenza viruses); Bungaviridae (e.g., Hantaan viruses, bunga viruses,phleboviruses and Nairo viruses); Arena viridae (hemorrhagic feverviruses); Reoviridae (e.g., reoviruses, orbiviurses and rotaviruses,i.e., Rotavirus A, Rotavirus B. Rotavirus C); Birnaviridae;Hepadnaviridae (Hepatitis A and B viruses); Parvoviridae (parvoviruses);Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (mostadenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, Humanherpes virus 6, Human herpes virus 7, Human herpes virus 8, varicellazoster virus, cytomegalovirus (CMV), herpes virus; Epstein-Barr virus;Rous sarcoma virus; West Nile virus; Japanese equine encephalitis,Norwalk, papilloma virus, parvovirus B19; Poxyiridae (variola viruses,vaccinia viruses, pox viruses); and Iridoviridae (e.g., African swinefever virus); Hepatitis D virus, Hepatitis E virus, and unclassifiedviruses (e.g., the etiological agents of Spongiform encephalopathies,the agent of delta hepatitis (thought to be a defective satellite ofhepatitis B virus), the agents of non-A, non-B hepatitis (class1=enterally transmitted; class 2=parenterally transmitted (i.e.,Hepatitis C); Norwalk and related viruses, and astroviruses).

Bacteria include both Gram negative and Gram positive bacteria. Examplesof Gram positive bacteria include, but are not limited to Pasteurellaspecies, Staphylococci species, and Streptococcus species. Examples ofGram negative bacteria include, but are not limited to, Escherichiacoli, Pseudomonas species, and Salmonella species. Specific examples ofinfectious bacteria include but are not limited to: Helicobacterpyloris, Borrelia burgdorferi, Legionella pneumophilia, Mycobacteriaspp. (e.g., M. tuberculosis, M. avium, M. intracellulare, M. kansasii,M. gordonae, M. leprae), Staphylococcus aureus, Neisseria gonorrhoeae,Neisseria meningitidis, Listeria monocytogenes, Streptococcus pyogenes(Group A Streptococcus), Streptococcus agalactiae (Group BStreptococcus), Streptococcus (viridans group), Streptococcus faecalis,Streptococcus bovis, Streptococcus (anaerobic spp.), Streptococcuspneumoniae, pathogenic Campylobacter spp., Enterococcus spp.,Haemophilus influenzae (Hemophilus influenza B, and Hemophilus influenzanon-typable), Bacillus anthracis, Corynebacterium diphtheriae,Corynebacterium spp., Erysipelothrix rhusiopathiae, Clostridiumperfringens, Clostridium tetani, Enterobacter aerogenes, Klebsiellapneumoniae, Pasturella multocida, Bacteroides spp., Fusobacteriumnucleatum, Streptobacillus moniliformis, Treponema pallidum, Treponemapertenue, Leptospira, Rickettsia, Actinomyces israelii, meningococcus,pertussis, pneumococcus, shigella, tetanus, Vibrio cholerae, yersinia,Pseudomonas species, Clostridia species, Salmonella typhi, Shigelladysenteriae, Yersinia pestis, Brucella species, Legionella pneumophila,Rickettsiae, Chlamydia, Clostridium perfringens, Clostridium botulinum,Staphylococcus aureus, Pseudomonas aeruginosa, Cryptosporidium parvum,Streptococcus pneumoniae, and Bordetella pertussis.

Exemplary fungi and yeast include, but are not limited to, Cryptococcusneoformans, Candida albicans, Candida tropicalis, Candida stellatoidea,Candida glabrata, Candida krusei, Candida parapsilosis, Candidaguilliermondii, Candida viswanathii, Candida lusitaniae, Rhodotorulamucilaginosa, Aspergillus fumigatus, Aspergillus flavus, Blastomycesdermatitidis, Aspergillus clavatus, Cryptococcus neoformans, Chlamydiatrachomatis, Coccidioides immitis, Cryptococcus laurentii, Cryptococcusalbidus, Cryptococcus gattii, Nocardia spp, Histoplasma capsulatum,Pneumocystis jirovecii (or Pneumocystis carinii), Stachybotryschartarum, and any combination thereof.

Exemplary parasites include, but are not limited to: Entamoebahistolytica; Plasmodium species (Plasmodium falciparum, Plasmodiummalariae, Plasmodium ovale, Plasmodium vivax), Leishmania species(Leishmania tropica, Leishmania braziliensis, Leishmania donovani),Toxoplasmosis (Toxoplasma gondii), Trypanosoma gambiense, Trypanosomarhodesiense (African sleeping sickness), Trypanosoma cruzi (Chagas'disease), Helminths (flat worms, round worms), Babesia microti, Babesiadivergens, Giardia lamblia, and any combination thereof.

The invention further relates to the use of an iRNA targeting one ormore EGLN genes and compositions containing at least one such iRNA forthe treatment of an infectious disease, such as hepatitis B or a chronicbacterial infection, in combination with other pharmaceuticals and/orother therapeutic methods, e.g., with known pharmaceuticals and/or knowntherapeutic methods, such as, for example, those which are currentlyemployed for treating such infectious diseases or disorders (e.g.,antibiotics, anti-viral agents). For example, in certain embodiments,administration of one or more dsRNA targeting EGLN is administered incombination with an antibacterial agent. Examples of anti-bacterialagents useful for the methods described herein include, but are notlimited to, natural penicillins, semi-synthetic penicillins, clavulanicacid, cephalolsporins, bacitracin, ampicillin, carbenicillin, oxacillin,azlocillin, mezlocillin, piperacillin, methicillin, dicloxacillin,nafcillin, cephalothin, cephapirin, cephalexin, cefamandole, cefaclor,cefazolin, cefuroxine, cefoxitin, cefotaxime, cefsulodin, cefetamet,cefixime, ceftriaxone, cefoperazone, ceftazidine, moxalactam,carbapenems, imipenems, monobactems, eurtreonam, vancomycin, polymyxin,amphotericin B, nystatin, imidazoles, clotrimazole, miconazole,ketoconazole, itraconazole, fluconazole, rifampins, ethambutol,tetracyclines, chloramphenicol, macrolides, aminoglycosides,streptomycin, kanamycin, tobramycin, amikacin, gentamicin, tetracycline,minocycline, doxycycline, chlortetracycline, erythromycin,roxithromycin, clarithromycin, oleandomycin, azithromycin,chloramphenicol, quinolones, co-trimoxazole, norfloxacin, ciprofloxacin,enoxacin, nalidixic acid, temafloxacin, sulfonamides, gantrisin, andtrimethoprim; Acedapsone; Acetosulfone Sodium; Alamecin; Alexidine;Amdinocillin; Amdinocillin Pivoxil; Amicycline; Amifloxacin; AmifloxacinMesylate; Amikacin; Amikacin Sulfate; Aminosalicylic acid;Aminosalicylate sodium; Amoxicillin; Amphomycin; Ampicillin; AmpicillinSodium; Apalcillin Sodium; Apramycin; Aspartocin; Astromicin Sulfate;Avilamycin; Avoparcin; Azithromycin; Azlocillin; Azlocillin Sodium;Bacampicillin Hydrochloride; Bacitracin; Bacitracin MethyleneDisalicylate; Bacitracin Zinc; Bambermycins; Benzoylpas Calcium;Berythromycin; Betamicin Sulfate; Biapenem; Biniramycin; BiphenamineHydrochloride; Bispyrithione Magsulfex; Butikacin; Butirosin Sulfate;Capreomycin Sulfate; Carbadox; Carbenicillin Disodium; CarbenicillinIndanyl Sodium; Carbenicillin Phenyl Sodium; Carbenicillin Potassium;Carumonam Sodium; Cefaclor; Cefadroxil; Cefamandole; Cefamandole Nafate;Cefamandole Sodium; Cefaparole; Cefatrizine; Cefazaflur Sodium;Cefazolin; Cefazolin Sodium; Cefbuperazone; Cefdinir; Cefepime; CefepimeHydrochloride; Cefetecol; Cefixime; Cefinenoxime Hydrochloride;Cefinetazole; Cefinetazole Sodium; Cefonicid Monosodium; CefonicidSodium; Cefoperazone Sodium; Ceforanide; Cefotaxime Sodium; Cefotetan;Cefotetan Disodium; Cefotiam Hydrochloride; Cefoxitin; Cefoxitin Sodium;Cefpimizole; Cefpimizole Sodium; Cefpiramide; Cefpiramide Sodium;Cefpirome Sulfate; Cefpodoxime Proxetil; Cefprozil; Cefroxadine;Cefsulodin Sodium; Ceftazidime; Ceftibuten; Ceftizoxime Sodium;Ceftriaxone Sodium; Cefuroxime; Cefuroxime Axetil; Cefuroxime Pivoxetil;Cefuroxime Sodium; Cephacetrile Sodium; Cephalexin; CephalexinHydrochloride; Cephaloglycin; Cephaloridine; Cephalothin Sodium;Cephapirin Sodium; Cephradine; Cetocycline Hydrochloride; Cetophenicol;Chloramphenicol; Chloramphenicol Palmitate; Chloramphenicol PantothenateComplex; Chloramphenicol Sodium Succinate; Chlorhexidine Phosphanilate;Chloroxylenol; Chlortetracycline Bisulfate; ChlortetracyclineHydrochloride; Cinoxacin; Ciprofloxacin; Ciprofloxacin Hydrochloride;Cirolemycin; Clarithromycin; Clinafloxacin Hydrochloride; Clindamycin;Clindamycin Hydrochloride; Clindamycin Palmitate Hydrochloride;Clindamycin Phosphate; Clofazimine; Cloxacillin Benzathine; CloxacillinSodium; Cloxyquin; Colistimethate Sodium; Colistin Sulfate; Coumermycin;Coumermycin Sodium; Cyclacillin; Cycloserine; Dalfopristin; Dapsone;Daptomycin; Demeclocycline; Demeclocycline Hydrochloride; Demecycline;Denofungin; Diaveridine; Dicloxacillin; Dicloxacillin Sodium;Dihydrostreptomycin Sulfate; Dipyrithione; Dirithromycin; Doxycycline;Doxycycline Calcium; Doxycycline Fosfatex; Doxycycline Hyclate; DroxacinSodium; Enoxacin; Epicillin; Epitetracycline Hydrochloride;Erythromycin; Erythromycin Acistrate; Erythromycin Estolate;Erythromycin Ethylsuccinate; Erythromycin Gluceptate; ErythromycinLactobionate; Erythromycin Propionate; Erythromycin Stearate; EthambutolHydrochloride; Ethionamide; Fleroxacin; Floxacillin; Fludalanine;Flumequine; Fosfomycin; Fosfomycin Tromethamine; Fumoxicillin;Furazolium Chloride; Furazolium Tartrate; Fusidate Sodium; Fusidic Acid;Gentamicin Sulfate; Gloximonam; Gramicidin; Haloprogin; Hetacillin;Hetacillin Potassium; Hexedine; Ibafloxacin; Inipenem; Isoconazole;Isepamicin; Isoniazid; Josamycin; Kanamycin Sulfate; Kitasamycin;Levofuraltadone; Levopropylcillin Potassium; Lexithromycin; Lincomycin;Lincomycin Hydrochloride; Lomefloxacin; Lomefloxacin Hydrochloride;Lomefloxacin Mesylate; Loracarbef; Mafenide; Meclocycline; MeclocyclineSulfosalicylate; Megalomicin Potassium Phosphate; Mequidox; Meropenem;Methacycline; Methacycline Hydrochloride; Methenamine; MethenamineHippurate; Methenamine Mandelate; Methicillin Sodium; Metioprim;Metronidazole Hydrochloride; Metronidazole Phosphate; Mezlocillin;Mezlocillin Sodium; Minocycline; Minocycline Hydrochloride; MirincamycinHydrochloride; Monensin; Monensin Sodium; Nafcillin Sodium; NalidixateSodium; Nalidixic Acid; Natamycin; Nebramycin; Neomycin Palmitate;Neomycin Sulfate; Neomycin Undecylenate; Netilmicin Sulfate;Neutramycin; Nifuradene; Nifuraldezone; Nifuratel; Nifuratrone;Nifurdazil; Nifurimide; Nifurpirinol; Nifurquinazol; Nifurthiazole;Nitrocycline; Nitrofurantoin; Nitromide; Norfloxacin; Novobiocin Sodium;Ofloxacin; Ormetoprim; Oxacillin Sodium; Oximonam; Oximonam Sodium;Oxolinic Acid; Oxytetracycline; Oxytetracycline Calcium; OxytetracyclineHydrochloride; Paldimycin; Parachlorophenol; Paulomycin; Pefloxacin;Pefloxacin Mesylate; Penamecillin; Penicillin G Benzathine; Penicillin GPotassium; Penicillin G Procaine; Penicillin G Sodium; Penicillin V;Penicillin V Benzathine; Penicillin V Hydrabamine; Penicillin VPotassium; Pentizidone Sodium; Phenyl Aminosalicylate; PiperacillinSodium; Pirbenicillin Sodium; Piridicillin Sodium; PirlimycinHydrochloride; Pivampicillin Hydrochloride; Pivampicillin Pamoate;Pivampicillin Probenate; Polymyxin B Sulfate; Porfiromycin; Propikacin;Pyrazinamide; Pyrithione Zinc; Quindecamine Acetate; Quinupristin;Racephenicol; Ramoplanin; Ranimycin; Relomycin; Repromicin; Rifabutin;Rifametane; Rifamexil; Rifamide; Rifampin; Rifapentine; Rifaximin;Rolitetracycline; Rolitetracycline Nitrate; Rosaramicin; RosaramicinButyrate; Rosaramicin Propionate; Rosaramicin Sodium Phosphate;Rosaramicin Stearate; Rosoxacin; Roxarsone; Roxithromycin; Sancycline;Sanfetrinem Sodium; Sarmoxicillin; Sarpicillin; Scopafungin; Sisomicin;Sisomicin Sulfate; Sparfloxacin; Spectinomycin Hydrochloride;Spiramycin; Stallimycin Hydrochloride; Steffimycin; StreptomycinSulfate; Streptonicozid; Sulfabenz; Sulfabenzamide; Sulfacetamide;Sulfacetamide Sodium; Sulfacytine; Sulfadiazine; Sulfadiazine Sodium;Sulfadoxine; Sulfalene; Sulfamerazine; Sulfameter; Sulfamethazine;Sulfamethizole; Sulfamethoxazole; Sulfamonomethoxine; Sulfamoxole;Sulfanilate Zinc; Sulfanitran; Sulfasalazine; Sulfasomizole;Sulfathiazole; Sulfazamet; Sulfisoxazole; Sulfisoxazole Acetyl;Sulfisoxazole Diolamine; Sulfomyxin; Sulopenem; Sultamicillin; SuncillinSodium; Talampicillin Hydrochloride; Teicoplanin; TemafloxacinHydrochloride; Temocillin; Tetracycline; Tetracycline Hydrochloride;Tetracycline Phosphate Complex; Tetroxoprim; Thiamphenicol;Thiphencillin Potassium; Ticarcillin Cresyl Sodium; TicarcillinDisodium; Ticarcillin Monosodium; Ticlatone; Tiodonium Chloride;Tobramycin; Tobramycin Sulfate; Tosufloxacin; Trimethoprim; TrimethoprimSulfate; Trisulfapyrimidines; Troleandomycin; Trospectomycin Sulfate;Tyrothricin; Vancomycin; Vancomycin Hydrochloride; Virginiamycin; andZorbamycin.

In other embodiments, administration of one or more dsRNA targeting oneor more EGLN genes is performed in combination with an anti-viralmedicament or agent. Exemplary antiviral agents useful for the methodsdescribed herein include, but are not limited to, immunoglobulins,amantadine, interferon, nucleoside analogues, and protease inhibitors.Specific examples of antiviral agents include but are not limited toAcemannan; Acyclovir; Acyclovir Sodium; Adefovir; Alovudine; AlvirceptSudotox; Amantadine Hydrochloride; Aranotin; Arildone; AtevirdineMesylate; Avridine; Cidofovir; Cipamfylline; Cytarabine Hydrochloride;Delavirdine Mesylate; Desciclovir; Didanosine; Disoxaril; Edoxudine;Enviradene; Enviroxime; Famciclovir; Famotine Hydrochloride;Fiacitabine; Fialuridine; Fosarilate; Foscamet Sodium; Fosfonet Sodium;Ganciclovir; Ganciclovir Sodium; Idoxuridine; Kethoxal; Lamivudine;Lobucavir; Memotine Hydrochloride; Methisazone; Nevirapine; Penciclovir;Pirodavir; Ribavirin; Rimantadine Hydrochloride; Saquinavir Mesylate;Somantadine Hydrochloride; Sorivudine; Statolon; Stavudine; TiloroneHydrochloride; Trifluridine; Valacyclovir Hydrochloride; Vidarabine;Vidarabine Phosphate; Vidarabine Sodium Phosphate; Viroxime;Zalcitabine; Zidovudine; and Zinviroxime.

In other embodiments, administration of one or more dsRNA targeting oneor more EGLN genes is performed in combination with an anti-fungalmedicament or agent. An “antifungal medicament” is an agent that killsor inhibits the growth or function of infective fungi. Anti-fungalmedicaments are sometimes classified by their mechanism of action. Someanti-fungal agents function as cell wall inhibitors by inhibitingglucose synthase, other antifungal agents function by destabilizingmembrane integrity, and other antifungal agents function by breakingdown chitin (e.g., chitinase) or immunosuppression (501 cream). Thus,exemplary antifungal medicaments useful for the methods described hereininclude, but are not limited to, imidazoles, 501 cream, and Acrisorcin,Ambruticin, Amorolfine, Amphotericin B, Azaconazole, Azaserine,Basifungin, BAY 38-9502, Bifonazole, Biphenamine Hydrochloride,Bispyrithione Magsulfex, Butenafine, Butoconazole Nitrate, CalciumUndecylenate, Candicidin, Carbol-Fuchsin, Chitinase, Chlordantoin,Ciclopirox, Ciclopirox Olamine, Cilofungin, Cisconazole, Clotrimazole,Cuprimyxin, Denofungin, Dipyrithione, Doconazole, Econazole, EconazoleNitrate, Enilconazole, Ethonam Nitrate, Fenticonazole Nitrate, Filipin,FK 463, Fluconazole, Flucytosine, Fungimycin, Griseofulvin, Hamycin,Isoconazole, Itraconazole, Kalafungin, Ketoconazole, Lomofungin,Lydimycin, Mepartricin, Miconazole, Miconazole Nitrate, MK 991,Monensin, Monensin Sodium, Naftifine Hydrochloride, NeomycinUndecylenate, Nifuratel, Nifurmerone, Nitralamine Hydrochloride,Nystatin, Octanoic Acid, Orconazole Nitrate, Oxiconazole Nitrate,Oxifungin Hydrochloride, Parconazole Hydrochloride, Partricin, PotassiumIodide, Pradimicin, Proclonol, Pyrithione Zinc, PyrroInitrin, Rutamycin,Sanguinarium Chloride, Saperconazole, Scopafungin, Selenium Sulfide,Sertaconazole, Sinefungin, Sulconazole Nitrate, Terbinafine,Terconazole, Thiram, Ticlatone, Tioconazole, Tolciclate, Tolindate,Tolnaftate, Triacetin, Triafungin, UK 292, Undecylenic Acid,Viridofulvin, Voriconazole, Zinc Undecylenate, and ZinoconazoleHydrochloride.

In further embodiments, administration of one or more dsRNA targetingone or more EGLN genes is administered in combination with ananti-parasitic medicament or agent. An “antiparasitic medicament” refersto an agent that kills or inhibits the growth or function of infectiveparasites. Examples of antiparasitic medicaments, also referred to asparasiticides, useful for the methods described herein include, but arenot limited to, albendazole, amphotericin B, benznidazole, bithionol,chloroquine HCl, chloroquine phosphate, clindamycin, dehydroemetine,diethylcarbamazine, diloxanide furoate, doxycycline, eflomithine,furazolidaone, glucocorticoids, halofantrine, iodoquinol, ivermectin,mebendazole, mefloquine, meglumine antimoniate, melarsoprol,metrifonate, metronidazole, niclosamide, nifurtimox, oxamniquine,paromomycin, pentamidine isethionate, piperazine, praziquantel,primaquine phosphate, proguanil, pyrantel pamoate,pyrimethanmine-sulfonamides, pyrimethanmine-sulfadoxine, quinacrine HCl,quinine sulfate, quinidine gluconate, spiramycin, stibogluconate sodium(sodium antimony gluconate), suramin, tetracycline, thiabendazole,timidazole, trimethroprim-sulfamethoxazole, and tryparsamide, some ofwhich are used alone or in combination with others.

The iRNA and an additional therapeutic agent can be administered incombination in the same composition, e.g., parenterally, or theadditional therapeutic agent can be administered as part of a separatecomposition or by another method described herein.

Patients can be administered a therapeutic amount of iRNA, such as 0.5mg/kg, 1.0 mg/kg, 1.5 mg/kg, 2.0 mg/kg, or 2.5 mg/kg dsRNA. The iRNA canbe administered by intravenous infusion over a period of time, such asover a 5 minute, 10 minute, 15 minute, 20 minute, or 25 minute period.The administration is repeated, for example, on a regular basis, such asbiweekly (i.e., every two weeks) for one month, two months, threemonths, four months or longer. After an initial treatment regimen, thetreatments can be administered on a less frequent basis. For example,after administration biweekly for three months, administration can berepeated once per month, for six months or a year or longer.Administration of the iRNA can reduce EGLN levels, e.g., in a cell,tissue, blood, urine or other compartment of the patient by at least10%, at least 15%, at least 20%, at least 25%, at least 30%, at least40%, at least 50%, at least 60%, at least 70%, at least 80% or at least90% or more.

Before administration of a full dose of the iRNA, patients can beadministered a smaller dose, such as a 5% infusion reaction, andmonitored for adverse effects, such as an allergic reaction, or forelevated lipid levels or blood pressure. In another example, the patientcan be monitored for unwanted immunostimulatory effects, such asincreased cytokine (e.g., TNF-alpha or INF-alpha) levels.

Genetic predisposition plays a role in the development of some cancersand hematological malignancies. Therefore, a patient in need of one ormore EGLN iRNA may be identified by taking a family history, or, forexample, screening for one or more genetic markers or variants. Ahealthcare provider, such as a doctor, nurse, or family member, can takea family history before prescribing or administering an EGLN dsRNA. Forexample, certain variants in the BRCA1 and BRCA2 genes are known tocause an increased risk for breast and ovarian cancers. A DNA test mayalso be performed on the patient to identify a mutation in an EGLN gene,before an EGLN dsRNA is administered to the patient.

Owing to the inhibitory effects on EGLN expression, a compositionaccording to the invention or a pharmaceutical composition preparedtherefrom can enhance the quality of life.

Methods for Modulating Expression of an EGLN Gene

In yet another aspect, the invention provides a method for modulating(e.g., inhibiting or activating) the expression of an EGLN gene in amammal

In one embodiment, the method includes administering a compositionfeatured in the invention to the mammal such that expression of thetarget EGLN gene is decreased, such as for an extended duration, e.g.,at least two, three, four days or more, e.g., one week, two weeks, threeweeks, or four weeks or longer.

In another embodiment, the method includes administering a compositionas described herein to a mammal such that expression of the target EGLNgene is increased by e.g., at least 10% compared to an untreated animal.In some embodiments, the activation of EGLN occurs over an extendedduration, e.g., at least two, three, four days or more, e.g., one week,two weeks, three weeks, four weeks, or more. Without wishing to be boundby theory, an iRNA can activate EGLN expression by stabilizing an EGLNmRNA transcript, interacting with a promoter in the genome, and/orinhibiting an inhibitor of EGLN expression.

Preferably, the iRNAs useful for the methods and compositions featuredin the invention specifically target RNAs (primary or processed) of thetarget EGLN gene. Compositions and methods for inhibiting the expressionof these EGLN genes using iRNAs can be prepared and performed asdescribed elsewhere herein.

In one embodiment, the method includes administering a compositioncontaining an iRNA, where the iRNA includes a nucleotide sequence thatis complementary to at least a part of an RNA transcript of an EGLN geneof the mammal to be treated. When the organism to be treated is a mammalsuch as a human, the composition may be administered by any means knownin the art including, but not limited to oral, intraperitoneal, orparenteral routes, including intracranial (e.g., intraventricular,intraparenchymal and intrathecal), intravenous, intramuscular,subcutaneous, transdermal, airway (aerosol), nasal, rectal, and topical(including buccal and sublingual) administration. In certainembodiments, the compositions are administered by intravenous infusionor injection.

In one embodiment iRNAs are able to substantially target a single organof the body. The targeted organ may be, but is not limited to, theliver, kidney and spleen. In another embodiment, the organ substantiallytargeted is the liver.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although methods and materialssimilar or equivalent to those described herein can be used in thepractice or testing of the iRNAs and methods featured in the invention,suitable methods and materials are described below. All publications,patent applications, patents, and other references mentioned herein areincorporated by reference in their entirety. In case of conflict, thepresent specification, including definitions, will control. In addition,the materials, methods, and examples are illustrative only and notintended to be limiting.

EXAMPLES Example 1 iRNA Synthesis

Source of Reagents

Where the source of a reagent is not specifically given herein, suchreagent may be obtained from any supplier of reagents for molecularbiology at a quality/purity standard for application in molecularbiology.

Oligonucleotide Synthesis.

All oligonucleotides are synthesized on an AKTAoligopilot synthesizer.Commercially available controlled pore glass solid support (dT-CPG, 500

, Prime Synthesis) and RNA phosphoramidites with standard protectinggroups, 5′-O-dimethoxytritylN6-benzoyl-2′-t-butyldimethylsilyl-adenosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N4-acetyl-2′-t-butyldimethylsilyl-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,5′-O-dimethoxytrityl-N2-isobutryl-2′-t-butyldimethylsilyl-guanosine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite,and5′-O-dimethoxytrityl-2′-t-butyldimethylsilyl-uridine-3′-O—N,N′-diisopropyl-2-cyanoethylphosphoramidite(Pierce Nucleic Acids Technologies) were used for the oligonucleotidesynthesis. The 2′-F phosphoramidites,5′-O-dimethoxytrityl-N4-acetyl-2′-fluoro-cytidine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeand5′-O-dimethoxytrityl-2′-fluoro-uridine-3′-O—N,N′-diisopropyl-2-cyanoethyl-phosphoramiditeare purchased from (Promega). All phosphoramidites are used at aconcentration of 0.2M in acetonitrile (CH₃CN) except for guanosine whichis used at 0.2M concentration in 10% THF/ANC (v/v). Coupling/recyclingtime of 16 minutes is used. The activator is 5-ethyl thiotetrazole(0.75M, American International Chemicals); for the PO-oxidationiodine/water/pyridine is used and for the PS-oxidation PADS (2%) in2,6-lutidine/ACN (1:1 v/v) is used.

3′-ligand conjugated strands are synthesized using solid supportcontaining the corresponding ligand. For example, the introduction ofcholesterol unit in the sequence is performed from ahydroxyprolinol-cholesterol phosphoramidite. Cholesterol is tethered totrans-4-hydroxyprolinol via a 6-aminohexanoate linkage to obtain ahydroxyprolinol-cholesterol moiety. 5′-end Cy-3 and Cy-5.5 (fluorophore)labeled iRNAs are synthesized from the corresponding Quasar-570 (Cy-3)phosphoramidite are purchased from Biosearch Technologies. Conjugationof ligands to 5′-end and or internal position is achieved by usingappropriately protected ligand-phosphoramidite building block. Anextended 15 min coupling of 0.1 M solution of phosphoramidite inanhydrous CH₃CN in the presence of 5-(ethylthio)-1H-tetrazole activatorto a solid-support-bound oligonucleotide. Oxidation of theinternucleotide phosphite to the phosphate is carried out using standardiodine-water as reported (1) or by treatment with tert-butylhydroperoxide/acetonitrile/water (10:87:3) with 10 min oxidation waittime conjugated oligonucleotide. Phosphorothioate is introduced by theoxidation of phosphite to phosphorothioate by using a sulfur transferreagent such as DDTT (purchased from AM Chemicals), PADS and or Beaucagereagent. The cholesterol phosphoramidite is synthesized in house andused at a concentration of 0.1 M in dichloromethane. Coupling time forthe cholesterol phosphoramidite is 16 minutes.

Deprotection I (Nucleobase Deprotection)

After completion of synthesis, the support is transferred to a 100 mLglass bottle (VWR). The oligonucleotide is cleaved from the support withsimultaneous deprotection of base and phosphate groups with 80 mL of amixture of ethanolic ammonia [ammonia:ethanol (3:1)] for 6.5 h at 55° C.The bottle is cooled briefly on ice and then the ethanolic ammoniamixture is filtered into a new 250-mL bottle. The CPG is washed with2×40 mL portions of ethanol/water (1:1 v/v). The volume of the mixtureis then reduced to ˜30 mL by roto-vap. The mixture is then frozen on dryice and dried under vacuum on a speed vac.

Deprotection II (Removal of 2′-TBDMS Group)

The dried residue is resuspended in 26 mL of triethylamine,triethylamine trihydrofluoride (TEA.3HF) or pyridine-HF and DMSO (3:4:6)and heated at 60° C. for 90 minutes to remove thetert-butyldimethylsilyl (TBDMS) groups at the 2′ position. The reactionis then quenched with 50 mL of 20 mM sodium acetate and the pH isadjusted to 6.5. Oligonucleotide is stored in a freezer untilpurification.

Analysis

The oligonucleotides are analyzed by high-performance liquidchromatography (HPLC) prior to purification and selection of buffer andcolumn depends on nature of the sequence and or conjugated ligand.

HPLC Purification

The ligand-conjugated oligonucleotides are purified by reverse-phasepreparative HPLC. The unconjugated oligonucleotides are purified byanion-exchange HPLC on a TSK gel column packed in house. The buffers are20 mM sodium phosphate (pH 8.5) in 10% CH₃CN (buffer A) and 20 mM sodiumphosphate (pH 8.5) in 10% CH₃CN, 1M NaBr (buffer B). Fractionscontaining full-length oligonucleotides are pooled, desalted, andlyophilized. Approximately 0.15 OD of desalted oligonucleotidess arediluted in water to 150 μL and then pipetted into special vials for CGEand LC/MS analysis. Compounds are then analyzed by LC-ESMS and CGE.

iRNA Preparation

For the general preparation of iRNA, equimolar amounts of sense andantisense strand are heated in 1×PBS at 95° C. for 5 min and slowlycooled to room temperature. Integrity of the duplex is confirmed by HPLCanalysis.

Nucleic acid sequences are represented below using standardnomenclature, and specifically the abbreviations of Table 1.

TABLE 1 Abbreviations of nucleotide monomers used in nucleic acidsequence representation. It will be understood that these monomers, whenpresent in an oligonucleotide, are mutually linked by5′-3′-phosphodiester bonds. Abbreviation Nucleotide(s) A Adenosine CCytidine G Guanosine T Thymidine U Uridine N any nucleotide (G, A, C, Tor U) a 2′-O-methyladenosine c 2′-O-methylcytidine g2′-O-methylguanosine u 2′-O-methyluridine dT 2′-deoxythymidine sphosphorothioate linkage

Example 2 EGLN siRNA Design and Synthesis Transcripts

Oligonucleotide design was carried out to identify siRNAs targeting thegenes encoding the mouse (Mus musculus) EGLN 1, 2 and 3 genes. Thedesign process used the EGLN transcript NM_(—)053207.2 for EGLN1 (SEQ IDNO: 5), NM_(—)053208.4 for EGLN2 (SEQ ID NO: 6), and NM_(—)028133.2 forEGLN3 (SEQ ID NO: 7). All sequences were obtained from the NCBI Refseqcollection.

The orthologous sequences from humans (Homo sapiens) were also designed.Oligonucleotide design was carried out to identify siRNAs targeting thegenes encoding the human (Homo sapiens) EGLN 1, 2 and 3 genes. Thedesign process used the EGLN transcript NM_(—)022051.2 for EGLN1 (SEQ IDNO: 390), NM_(—)053046.2 for EGLN2 (SEQ ID NO: 391), and NM_(—)022073.3for EGLN3 (SEQ ID NO: 392). All sequences were obtained from the NCBIRefseq collection.

The set of mouse EGLN derived siRNA oligos designed and synthesized arepresented in Tables 2A-F.

The set of human EGLN derived siRNA oligonucleotide single and doublestrand duplexes designed are presented in Tables 6A-C.

siRNA Design and Specificity Prediction

The specificity of the 19mer oligo sets was predicted from eachsequence. The EGLN siRNAs were used in a comprehensive search againsttheir respective human, or mouse and rat transcriptomes (defined as theset of NM_ and XM_records within the NCBI Refseq set) using the FASTAalgorithm. The Python script ‘offtargetFasta.py’ was then used to parsethe alignments and generate a score based on the position and number ofmismatches between the siRNA and any potential ‘off-target’ transcript.The off-target score is weighted to emphasize differences in the ‘seed’region of siRNAs, in positions 2-9 from the 5′ end of the molecule. Theoff-target score is calculated as follows: mismatches between the oligoand the transcript are given penalties. A mismatch in the seed region inpositions 2-9 of the oligo is given a penalty of 2.8; mismatches in theputative cleavage sites 10 and 11 are given a penalty of 1.2, and allother mismatches a penalty of 1. The off-target score for eacholigo-transcript pair is then calculated by summing the mismatchpenalties. The lowest off-target score from all the oligo-transcriptpairs is then determined and used in subsequent sorting of oligos. BothsiRNAs strands were assigned to a category of specificity according tothe calculated scores: a score above 3 qualifies as highly specific,equal to 3 as specific and between 2.2 and 2.8 as moderate specific. Inpicking which oligos to synthesize, we sorted from high to low by theoff-target score of the antisense strand and took the best (lowestoff-target score) oligo pairs.

Synthesis of EGLN Sequences

EGLN targeting sequences were synthesized on a MerMade 192 synthesizerat 1 μmol scale.

For all chemically modified sequences in the list, ‘endolight’ chemistrywas applied as detailed below.

-   -   All pyrimidines (cytosine and uridine) in the sense strand        contained 2′-O-Methyl bases (2′ O-Methyl C and 2′-O-Methyl U)    -   In the antisense strand, pyrimidines adjacent to (towards 5′        position) ribo A nucleoside were replaced with their        corresponding 2-O-Methyl nucleosides    -   A two base dTsdT extension at 3′ end of both sense and antisense        sequences was introduced    -   The sequence file was converted to a text file to make it        compatible for loading in the MerMade 192 synthesis software

Synthesis, Cleavage and Deprotection:

The synthesis of EGLN sequences used solid supported oligonucleotidesynthesis using phosphoramidite chemistry.

The synthesis of the above sequences was performed at 1 um scale in 96well plates. The amidite solutions were prepared at 0.1M concentrationand ethyl thio tetrazole (0.6M in Acetonitrile) was used as activator.

The synthesized sequences were cleaved and deprotected in 96 wellplates, using methylamine in the first step and fluoride reagent in thesecond step. The crude sequences were precipitated using acetone:ethanol(80:20) mix and the pellet were re-suspended in 0.02M sodium acetatebuffer. Samples from each sequence were analyzed by LC-MS to confirm theidentity, UV for quantification and a selected set of samples by IEXchromatography to determine purity.

Purification and Desalting:

EGLN sequences were purified on AKTA explorer purification system usingSource 15Q column. A column temperature of 65 C was maintained duringpurification. Sample injection and collection was performed in 96 well(1.8 mL-deep well) plates. A single peak corresponding to the fulllength sequence was collected in the eluent. The purified sequences weredesalted on a Sephadex G25 column using AKTA purifier. The desalted EGLNsequences were analyzed for concentration (by UV measurement at A260)and purity (by ion exchange HPLC). The single strands were thensubmitted for annealing. The control duplex, AD-1955, which targets theluciferase gene has the sense sequence cuuAcGcuGAGuAcuucGAdTsdT (SEQ IDNO: 8) and the antisense sequence UCGAAGuACUcAGCGuAAGdTsdT (SEQ ID NO:9), where lower case nucleotides are modified by 2′Omethyl and dT standsfor deoxyThymidine and “s” represents a phosphorothioate linkage.

TABLE 2A  Mouse EGNL1 Single Strands and Duplex Sequences SEQ SEQ DuplexSequence (5′ to 3′) ID Sequence (5′ to 3′) ID Number Start* Sense NO.Antisense NO. AD-40893 1057 GCUAUGUCCGUCACGUUGA 10 UCAACGUGACGGACAUAGC11 AD-40899 1065 CGUCACGUUGAUAACCCAA 12 UUGGGUUAUCAACGUGACG 13 AD-409051092 GGAAGAUGCGUGACAUGUA 14 UACAUGUCACGCAUCUUCC 15 AD-40911 1128GACUGGGACGCCAAGGUAA 16 UUACCUUGGCGUCCCAGUC 17 AD-40917 1150GAGGUAUUCUUCGAAUUUU 18 AAAAUUCGAAGAAUACCUC 19 AD-40923 1240GGCGUAACCCUCAUGAAGU 20 ACUUCAUGAGGGUUACGCC 21 AD-40929 1271CGCCACAAGGUACGCAAUA 22 UAUUGCGUACCUUGUGGCG 23 AD-40888 1272GCCACAAGGUACGCAAUAA 24 UUAUUGCGUACCUUGUGGC 25 AD-40894 1276CAAGGUACGCAAUAACUGU 26 ACAGUUAUUGCGUACCUUG 27 AD-40900 1317CGAGCGAGAGCUAAAGUAA 28 UUACUUUAGCUCUCGCUCG 29 AD-40906 1320GCGAGAGCUAAAGUAAAAU 30 AUUUUACUUUAGCUCUCGC 31 AD-40912 1356GGUGUGAGGGUUGAACUCA 32 UGAGUUCAACCCUCACACC 33 AD-40918 1386GUCAGCAAAGACGUCUAGU 34 ACUAGACGUCUUUGCUGAC 35 AD-40924 1892GCCUCCUGCGAUGAUUGUU 36 AACAAUCAUCGCAGGAGGC 37 AD-40930 1919GUGACGACGUGUUGCUUCU 38 AGAAGCAACACGUCGUCAC 39 AD-40889 2043CGCUUCGACCGACCUAACA 40 UGUUAGGUCGGUCGAAGCG 41 AD-40895 2048CGACCGACCUAACAGUAGA 42 UCUACUGUUAGGUCGGUCG 43 AD-40901 2093CAACAUAGUUACAAGAGGA 44 UCCUCUUGUAACUAUGUUG 45 AD-40907 2159CGAAGUGACGGGCACUAAA 46 UUUAGUGCCCGUCACUUCG 47 AD-40913 2160GAAGUGACGGGCACUAAAU 48 AUUUAGUGCCCGUCACUUC 49 AD-40919 2372GUGAAUGUGGUAUGUGGUU 50 AACCACAUACCACAUUCAC 51 AD-40925 2605GCACAGAUUGUGGGUAUAA 52 UUAUACCCACAAUCUGUGC 53 AD-40931 2624CUCCUGUCCCCUUAGGUGU 54 ACACCUAAGGGGACAGGAG 55 AD-40890 2732GUUUGUAUCCGGUUAGAAA 56 UUUCUAACCGGAUACAAAC 57 AD-40896 2889GUCUCCUUCUGACCCAUAU 58 AUAUGGGUCAGAAGGAGAC 59 AD-40902 2894CUUCUGACCCAUAUCCGCU 60 AGCGGAUAUGGGUCAGAAG 61 AD-40908 3001GGAACUGUUUGGCAUUGUU 62 AACAAUGCCAAACAGUUCC 63 AD-40914 3244CUUAAUGCCCACUUAAACU 64 AGUUUAAGUGGGCAUUAAG 65 AD-40920 3272GUUAGGACUCUUGUUUAAA 66 UUUAAACAAGAGUCCUAAC 67 AD-40926 3350CUGUUCAACACAUUAACCA 68 UGGUUAAUGUGUUGAACAG 69 AD-40932 3472GCUUGUAAAGCUAAUCUAA 70 UUAGAUUAGCUUUACAAGC 71 *Start is the 5′ mostposition on transcript NM_053207.2

TABLE 2B Mouse EGNL1 Chemically modified Single Strands and Duplex SequencesFor all the sequences in the list, ‘endolight’ chemistry was applied asdescribed above. SEQ SEQ Duplex Sequence (5′ to 3′) ID Sequence (5′to 3′) ID Number Start* Sense NO. Antisense NO. AD-40893 1057GcuAuGuccGucAcGuuGAdTsdT 72 UcAACGUGACGGAcAuAGCdTsdT 73 AD-40899 1065cGucAcGuuGAuAAcccAAdTsdT 74 UUGGGUuAUcAACGUGACGdTsdT 75 AD-40905 1092GGAAGAuGcGuGAcAuGuAdTsdT 76 uAcAUGUcACGcAUCUUCCdTsdT 77 AD-40911 1128GAcuGGGAcGccAAGGuAAdTsdT 78 UuACCUUGGCGUCCcAGUCdTsdT 79 AD-40917 1150GAGGuAuucuucGAAuuuudTsdT 80 AAAAUUCGAAGAAuACCUCdTsdT 81 AD-40923 1240GGcGuAAcccucAuGAAGudTsdT 82 ACUUcAUGAGGGUuACGCCdTsdT 83 AD-40929 1271cGccAcAAGGuAcGcAAuAdTsdT 84 uAUUGCGuACCUUGUGGCGdTsdT 85 AD-40888 1272GccAcAAGGuAcGcAAuAAdTsdT 86 UuAUUGCGuACCUUGUGGCdTsdT 87 AD-40894 1276cAAGGuAcGcAAuAAcuGudTsdT 88 AcAGUuAUUGCGuACCUUGdTsdT 89 AD-40900 1317cGAGcGAGAGcuAAAGuAAdTsdT 90 UuACUUuAGCUCUCGCUCGdTsdT 91 AD-40906 1320GcGAGAGcuAAAGuAAAAudTsdT 92 AUUUuACUUuAGCUCUCGCdTsdT 93 AD-40912 1356GGuGuGAGGGuuGAAcucAdTsdT 94 UGAGUUcAACCCUcAcACCdTsdT 95 AD-40918 1386GucAGcAAAGAcGucuAGudTsdT 96 ACuAGACGUCUUUGCUGACdTsdT 97 AD-40924 1892GccuccuGcGAuGAuuGuudTsdT 98 AAcAAUcAUCGcAGGAGGCdTsdT 99 AD-40930 1919GuGAcGAcGuGuuGcuucudTsdT 100 AGAAGcAAcACGUCGUcACdTsdT 101 AD-40889 2043cGcuucGAccGAccuAAcAdTsdT 102 UGUuAGGUCGGUCGAAGCGdTsdT 103 AD-40895 2048cGAccGAccuAAcAGuAGAdTsdT 104 UCuACUGUuAGGUCGGUCGdTsdT 105 AD-40901 2093cAAcAuAGuuAcAAGAGGAdTsdT 106 UCCUCUUGuAACuAUGUUGdTsdT 107 AD-40907 2159cGAAGuGAcGGGcAcuAAAdTsdT 108 UUuAGUGCCCGUcACUUCGdTsdT 109 AD-40913 2160GAAGuGAcGGGcAcuAAAudTsdT 110 AUUuAGUGCCCGUcACUUCdTsdT 111 AD-40919 2372GuGAAuGuGGuAuGuGGuudTsdT 112 AACcAcAuACcAcAUUcACdTsdT 113 AD-40925 2605GcAcAGAuuGuGGGuAuAAdTsdT 114 UuAuACCcAcAAUCUGUGCdTsdT 115 AD-40931 2624cuccuGuccccuuAGGuGudTsdT 116 AcACCuAAGGGGAcAGGAGdTsdT 117 AD-40890 2732GuuuGuAuccGGuuAGAAAdTsdT 118 UUUCuAACCGGAuAcAAACdTsdT 119 AD-40896 2889GucuccuucuGAcccAuAudTsdT 120 AuAUGGGUcAGAAGGAGACdTsdT 121 AD-40902 2894cuucuGAcccAuAuccGcudTsdT 122 AGCGGAuAUGGGUcAGAAGdTsdT 123 AD-40908 3001GGAAcuGuuuGGcAuuGuudTsdT 124 AAcAAUGCcAAAcAGUUCCdTsdT 125 AD-40914 3244cuuAAuGcccAcuuAAAcudTsdT 126 AGUUuAAGUGGGcAUuAAGdTsdT 127 AD-40920 3272GuuAGGAcucuuGuuuAAAdTsdT 128 UUuAAAcAAGAGUCCuAACdTsdT 129 AD-40926 3350cuGuucAAcAcAuuAAccAdTsdT 130 UGGUuAAUGUGUUGAAcAGdTsdT 131 AD-40932 3472GcuuGuAAAGcuAAucuAAdTsdT 132 UuAGAUuAGCUUuAcAAGCdTsdT 133

TABLE 2C Mouse EGNL2 Single Strands and Duplex Sequences SEQ SEQ DuplexSequence (5′ to 3′) ID Sequence (5′ to 3′) ID Number Start* Sense NO.Antisense NO. AD- 128 AUCAGUCCCUUCUCAAGCU 134 AGCUUGAGAAGGGACUGAU 13540891 AD- 418 GUCCUUGGAGUCUAGCCGA 136 UCGGCUAGACUCCAAGGAC 137 40897 AD-545 GCCACUGCUACUACGACCA 138 UGGUCGUAGUAGCAGUGGC 139 40903 AD- 934GCCUUGCAUGCGGUACUAU 140 AUAGUACCGCAUGCAAGGC 141 40909 AD- 941AUGCGGUACUAUGGUAUCU 142 AGAUACCAUAGUACCGCAU 143 40915 AD- 943GCGGUACUAUGGUAUCUGU 144 ACAGAUACCAUAGUACCGC 145 40921 AD- 956AUCUGUGUCAAGGACAACU 146 AGUUGUCCUUGACACAGAU 147 40927 AD- 1043CGUGAUGGGCAACUAGUGA 148 UCACUAGUUGCCCAUCACG 149 40933 AD- 1107CCUGGGUAGAAGGUCACGA 150 UCGUGACCUUCUACCCAGG 151 40892 AD- 1158CUCACGUGGACGCAGUAAU 152 AUUACUGCGUCCACGUGAG 153 40898 AD- 1228GGCCAUGGUGGCGUGUUAU 154 AUAACACGCCACCAUGGCC 155 40904 AD- 1235GUGGCGUGUUAUCCAGGCA 156 UGCCUGGAUAACACGCCAC 157 40910 AD- 1253AAUGGGCUCGGGUACGUGA 158 UCACGUACCCGAGCCCAUU 159 40916 AD- 1261CGGGUACGUGAGGCAUGUU 160 AACAUGCCUCACGUACCCG 161 40922 AD- 1263GGUACGUGAGGCAUGUUGA 162 UCAACAUGCCUCACGUACC 163 40928 AD- 1272GGCAUGUUGACAAUCCCCA 164 UGGGGAUUGUCAACAUGCC 165 40934 AD- 1305GCAUCACCUGUAUCUAUUA 166 UAAUAGAUACAGGUGAUGC 167 40743 AD- 1329AUCAGAACUGGGAUGUUAA 168 UUAACAUCCCAGUUCUGAU 169 40749 AD- 1335ACUGGGAUGUUAAGGUGCA 170 UGCACCUUAACAUCCCAGU 171 40755 AD- 1399CAACAUCGAGCCACUCUUU 172 AAAGAGUGGCUCGAUGUUG 173 40761 AD- 1534CAGAGACAAGUAUCAGCUA 174 UAGCUGAUACUUGUCUCUG 175 40767 AD- 1537AGACAAGUAUCAGCUAGCA 176 UGCUAGCUGAUACUUGUCU 177 40773 AD- 1555AUCGGGACAGAAAGGUGUU 178 AACACCUUUCUGUCCCGAU 179 40779 AD- 1567AGGUGUUCAAGUACCAGUA 180 UACUGGUACUUGAACACCU 181 40785 AD- 1708GUGGUGUGGAGGGCACUAA 182 UUAGUGCCCUCCACACCAC 183 40744 AD- 1710GGUGUGGAGGGCACUAAGU 184 ACUUAGUGCCCUCCACACC 185 40750 AD- 1711GUGUGGAGGGCACUAAGUA 186 UACUUAGUGCCCUCCACAC 187 40756 AD- 1830UGGCUGUGUCUGGUCCGUU 188 AACGGACCAGACACAGCCA 189 40762 AD- 1872GGAUUUGGGGUUGAGGUGA 190 UCACCUCAACCCCAAAUCC 191 40768 AD- 1876UUGGGGUUGAGGUGAGUCA 192 UGACUCACCUCAACCCCAA 193 40774 AD- 1917GUUGGGGUGUGGGUGUCAU 194 AUGACACCCACACCCCAAC 195 40780 AD- 2038AGGGUGCCAUGACGAGCAU 196 AUGCUCGUCAUGGCACCCU 197 40786 *Start is the 5′most position on transcript NM_053208.4

TABLE 2D Mouse EGNL2 Chemically modified Single Strands and DuplexSequences For all the sequences in the list, ‘endolight’chemistry was applied as described above. SEQ SEQ Duplex Sequence (5′to 3′) ID Sequence (5′ to 3′) ID Number Start* Sense NO. Antisense NO.AD-40891 128 AucAGucccuucucAAGcudTsdT 198 AGCUUGAGAAGGGACUGAUdTsdT 199AD-40897 418 GuccuuGGAGucuAGccGAdTsdT 200 UCGGCuAGACUCcAAGGACdTsdT 201AD-40903 545 GccAcuGcuAcuAcGAccAdTsdT 202 UGGUCGuAGuAGcAGUGGCdTsdT 203AD-40909 934 GccuuGcAuGcGGuAcuAudTsdT 204 AuAGuACCGcAUGcAAGGCdTsdT 205AD-40915 941 AuGcGGuAcuAuGGuAucudTsdT 206 AGAuACcAuAGuACCGcAUdTsdT 207AD-40921 943 GcGGuAcuAuGGuAucuGudTsdT 208 AcAGAuACcAuAGuACCGCdTsdT 209AD-40927 956 AucuGuGucAAGGAcAAcudTsdT 210 AGUUGUCCUUGAcAcAGAUdTsdT 211AD-40933 1043 cGuGAuGGGcAAcuAGuGAdTsdT 212 UcACuAGUUGCCcAUcACGdTsdT 213AD-40892 1107 ccuGGGuAGAAGGucAcGAdTsdT 214 UCGUGACCUUCuACCcAGGdTsdT 215AD-40898 1158 cucAcGuGGAcGcAGuAAudTsdT 216 AUuACUGCGUCcACGUGAGdTsdT 217AD-40904 1228 GGccAuGGuGGcGuGuuAudTsdT 218 AuAAcACGCcACcAUGGCCdTsdT 219AD-40910 1235 GuGGcGuGuuAuccAGGcAdTsdT 220 UGCCUGGAuAAcACGCcACdTsdT 221AD-40916 1253 AAuGGGcucGGGuAcGuGAdTsdT 222 UcACGuACCCGAGCCcAUUdTsdT 223AD-40922 1261 cGGGuAcGuGAGGcAuGuudTsdT 224 AAcAUGCCUcACGuACCCGdTsdT 225AD-40928 1263 GGuAcGuGAGGcAuGuuGAdTsdT 226 UcAAcAUGCCUcACGuACCdTsdT 227AD-40934 1272 GGcAuGuuGAcAAuccccAdTsdT 228 UGGGGAUUGUcAAcAUGCCdTsdT 229AD-40743 1305 GcAucAccuGuAucuAuuAdTsdT 230 uAAuAGAuAcAGGUGAUGCdTsdT 231AD-40749 1329 AucAGAAcuGGGAuGuuAAdTsdT 232 UuAAcAUCCcAGUUCUGAUdTsdT 233AD-40755 1335 AcuGGGAuGuuAAGGuGcAdTsdT 234 UGcACCUuAAcAUCCcAGUdTsdT 235AD-40761 1399 cAAcAucGAGccAcucuuudTsdT 236 AAAGAGUGGCUCGAUGUUGdTsdT 237AD-40767 1534 cAGAGAcAAGuAucAGcuAdTsdT 238 uAGCUGAuACUUGUCUCUGdTsdT 239AD-40773 1537 AGAcAAGuAucAGcuAGcAdTsdT 240 UGCuAGCUGAuACUUGUCUdTsdT 241AD-40779 1555 AucGGGAcAGAAAGGuGuudTsdT 242 AAcACCUUUCUGUCCCGAUdTsdT 234AD-40785 1567 AGGuGuucAAGuAccAGuAdTsdT 244 uACUGGuACUUGAAcACCUdTsdT 245AD-40744 1708 GuGGuGuGGAGGGcAcuAAdTsdT 246 UuAGUGCCCUCcAcACcACdTsdT 247AD-40750 1710 GGuGuGGAGGGcAcuAAGudTsdT 248 ACUuAGUGCCCUCcAcACCdTsdT 249AD-40756 1711 GuGuGGAGGGcAcuAAGuAdTsdT 250 uACUuAGUGCCCUCcAcACdTsdT 251AD-40762 1830 uGGcuGuGucuGGuccGuudTsdT 252 AACGGACcAGAcAcAGCcAdTsdT 253AD-40768 1872 GGAuuuGGGGuuGAGGuGAdTsdT 254 UcACCUcAACCCcAAAUCCdTsdT 255AD-40774 1876 uuGGGGuuGAGGuGAGucAdTsdT 256 UGACUcACCUcAACCCcAAdTsdT 257AD-40780 1917 GuuGGGGuGuGGGuGucAudTsdT 258 AUGAcACCcAcACCCcAACdTsdT 259AD-40786 2038 AGGGuGccAuGAcGAGcAudTsdT 260 AUGCUCGUcAUGGcACCCUdTsdT 261

TABLE 2E Mouse EGNL3 Single Strands and Duplex Sequences SEQ SEQ DuplexSequence (5′ to 3′) ID Sequence (5′ to 3′) ID Number Start* Sense NO.Antisense NO. AD- 634 CCGGCUGGGCAAAUACUAU 262 AUAGUAUUUGCCCAGCCGG 26340745 AD- 775 GAAUUGGGACGCCAAGUUA 264 UAACUUGGCGUCCCAAUUC 265 40751 AD-819 CGGAAGGGAAAUCGUUUGU 266 ACAAACGAUUUCCCUUCCG 267 40757 AD- 882CAGACCGCAGGAAUCCACA 268 UGUGGAUUCCUGCGGUCUG 269 40763 AD- 922CACCAGGUACGCUAUGACU 270 AGUCAUAGCGUACCUGGUG 271 40769 AD- 924CCAGGUACGCUAUGACUGU 272 ACAGUCAUAGCGUACCUGG 273 40775 AD- 937GACUGUCUGGUACUUCGAU 274 AUCGAAGUACCAGACAGUC 275 40781 AD- 1053GGCCGCAUUCGUGUAGUAA 276 UUACUACACGAAUGCGGCC 277 40787 AD- 1055CCGCAUUCGUGUAGUAACA 278 UGUUACUACACGAAUGCGG 279 40746 AD- 1058CAUUCGUGUAGUAACAGUU 280 AACUGUUACUACACGAAUG 281 40752 AD- 1065GUAGUAACAGUUCCGGAAA 282 UUUCCGGAACUGUUACUAC 283 40758 AD- 1068GUAACAGUUCCGGAAAUGU 284 ACAUUUCCGGAACUGUUAC 285 40764 AD- 1265CCAGCGGUUUAAAGAUAGA 286 UCUAUCUUUAAACCGCUGG 287 40770 AD- 1309GGACUGCUUCUUAUUCGCA 288 UGCGAAUAAGAAGCAGUCC 289 40776 AD- 1312CUGCUUCUUAUUCGCACUU 290 AAGUGCGAAUAAGAAGCAG 291 40782 AD- 1318CUUAUUCGCACUUUAUGUA 292 UACAUAAAGUGCGAAUAAG 293 40788 AD- 1334GUAUGCGUCCUGAUUUGAA 294 UUCAAAUCAGGACGCAUAC 295 40747 AD- 1358GAGGUUCGCAAAGAAAUAA 296 UUAUUUCUUUGCGAACCUC 297 40753 AD- 1474GACAGUGACGACGACCUAA 298 UUAGGUCGUCGUCACUGUC 299 40759 AD- 1480GACGACGACCUAAUGACAU 300 AUGUCAUUAGGUCGUCGUC 301 40765 AD- 1482CGACGACCUAAUGACAUUA 302 UAAUGUCAUUAGGUCGUCG 303 40771 AD- 1516GCUGCUGCUUAGCAAUCGA 304 UCGAUUGCUAAGCAGCAGC 305 40777 AD- 1517CUGCUGCUUAGCAAUCGAU 306 AUCGAUUGCUAAGCAGCAG 307 40783 AD- 1548CACGGUGGAUGCUCCAUUU 308 AAAUGGAGCAUCCACCGUG 309 40789 AD- 1571GGUUUACGACCCGUACUUU 310 AAAGUACGGGUCGUAAACC 311 40748 AD- 1815CCCAACUUACAUGAUUCGU 312 ACGAAUCAUGUAAGUUGGG 313 40754 AD- 1929GUUCAUCGUCCAUAACAAA 314 UUUGUUAUGGACGAUGAAC 315 40760 AD- 2034CUCACUUGAGUCGUCUUGA 316 UCAAGACGACUCAAGUGAG 317 40766 AD- 2146CCUCCCGAACUCUGUACGA 318 UCGUACAGAGUUCGGGAGG 319 40772 AD- 2157CUGUACGAAACACCUAUUU 320 AAAUAGGUGUUUCGUACAG 321 40778 AD- 2162CGAAACACCUAUUUUACGA 322 UCGUAAAAUAGGUGUUUCG 323 40784 AD- 2163GAAACACCUAUUUUACGAA 324 UUCGUAAAAUAGGUGUUUC 325 40790 *Start is the 5′most position on transcript NM_028133.2

TABLE 2F Mouse EGNL3 Chemically modified Single Strands and DuplexSequences For all the sequences in the list, ‘endolight’chemistry was applied as described above. SEQ SEQ Duplex Sequence (5′to 3′) ID Sequence (5′ to 3′) ID Number Start* Sense NO. Antisense NO.AD-40745 634 ccGGcuGGGcAAAuAcuAudTsdT 326 AuAGuAUUUGCCcAGCCGGdTsdT 327AD-40751 775 GAAuuGGGAcGccAAGuuAdTsdT 328 uAACUUGGCGUCCcAAUUCdTsdT 329AD-40757 819 cGGAAGGGAAAucGuuuGudTsdT 330 AcAAACGAUUUCCCUUCCGdTsdT 331AD-40763 882 cAGAccGcAGGAAuccAcAdTsdT 332 UGUGGAUUCCUGCGGUCUGdTsdT 333AD-40769 922 cAccAGGuAcGcuAuGAcudTsdT 334 AGUcAuAGCGuACCUGGUGdTsdT 335AD-40775 924 ccAGGuAcGcuAuGAcuGudTsdT 336 AcAGUcAuAGCGuACCUGGdTsdT 337AD-40781 937 GAcuGucuGGuAcuucGAudTsdT 338 AUCGAAGuACcAGAcAGUCdTsdT 339AD-40787 1053 GGccGcAuucGuGuAGuAAdTsdT 340 UuACuAcACGAAUGCGGCCdTsdT 341AD-40746 1055 ccGcAuucGuGuAGuAAcAdTsdT 342 UGUuACuAcACGAAUGCGGdTsdT 343AD-40752 1058 cAuucGuGuAGuAAcAGuudTsdT 344 AACUGUuACuAcACGAAUGdTsdT 345AD-40758 1065 GuAGuAAcAGuuccGGAAAdTsdT 346 UUUCCGGAACUGUuACuACdTsdT 347AD-40764 1068 GuAAcAGuuccGGAAAuGudTsdT 348 AcAUUUCCGGAACUGUuACdTsdT 349AD-40770 1265 ccAGcGGuuuAAAGAuAGAdTsdT 350 UCuAUCUUuAAACCGCUGGdTsdT 351AD-40776 1309 GGAcuGcuucuuAuucGcAdTsdT 352 UGCGAAuAAGAAGcAGUCCdTsdT 353AD-40782 1312 cuGcuucuuAuucGcAcuudTsdT 354 AAGUGCGAAuAAGAAGcAGdTsdT 355AD-40788 1318 cuuAuucGcAcuuuAuGuAdTsdT 356 uAcAuAAAGUGCGAAuAAGdTsdT 357AD-40747 1334 GuAuGcGuccuGAuuuGAAdTsdT 358 UUcAAAUcAGGACGcAuACdTsdT 359AD-40753 1358 GAGGuucGcAAAGAAAuAAdTsdT 360 UuAUUUCUUUGCGAACCUCdTsdT 361AD-40759 1474 GAcAGuGAcGAcGAccuAAdTsdT 362 UuAGGUCGUCGUcACUGUCdTsdT 363AD-40765 1480 GAcGAcGAccuAAuGAcAudTsdT 364 AUGUcAUuAGGUCGUCGUCdTsdT 365AD-40771 1482 cGAcGAccuAAuGAcAuuAdTsdT 366 uAAUGUcAUuAGGUCGUCGdTsdT 367AD-40777 1516 GcuGcuGcuuAGcAAucGAdTsdT 368 UCGAUUGCuAAGcAGcAGCdTsdT 369AD-40783 1517 cuGcuGcuuAGcAAucGAudTsdT 370 AUCGAUUGCuAAGcAGcAGdTsdT 371AD-40789 1548 cAcGGuGGAuGcuccAuuudTsdT 372 AAAUGGAGcAUCcACCGUGdTsdT 373AD-40748 1571 GGuuuAcGAcccGuAcuuudTsdT 374 AAAGuACGGGUCGuAAACCdTsdT 375AD-40754 1815 cccAAcuuAcAuGAuucGudTsdT 376 ACGAAUcAUGuAAGUUGGGdTsdT 377AD-40760 1929 GuucAucGuccAuAAcAAAdTsdT 378 UUUGUuAUGGACGAUGAACdTsdT 379AD-40766 2034 cucAcuuGAGucGucuuGAdTsdT 380 UcAAGACGACUcAAGUGAGdTsdT 381AD-40772 2146 ccucccGAAcucuGuAcGAdTsdT 382 UCGuAcAGAGUUCGGGAGGdTsdT 383AD-40778 2157 cuGuAcGAAAcAccuAuuudTsdT 384 AAAuAGGUGUUUCGuAcAGdTsdT 385AD-40784 2162 cGAAAcAccuAuuuuAcGAdTsdT 386 UCGuAAAAuAGGUGUUUCGdTsdT 387AD-40790 2163 GAAAcAccuAuuuuAcGAAdTsdT 388 UUCGuAAAAuAGGUGUUUCdTsdT 389RNA Isolation, cDNA Synthesis and RT-PCR MethodsTotal RNA isolation using MagMAX-96 Total RNA Isolation Kit (AppliedBiosystem, Forer City Calif., part #: AM1830):

Cells were harvested and lysed in 140 μl of Lysis/Binding Solution thenmixed for 1 minute at 850 rpm using and Eppendorf Thermomixer (themixing speed was the same throughout the process). Twenty micro litersof magnetic beads and Lysis/Binding Enhancer mixture were added intocell-lysate and mixed for 5 minutes. Magnetic beads were captured usingmagnetic stand and the supernatant was removed without disturbing thebeads. After removing supernatant, magnetic beads were washed with WashSolution 1 (isopropanol added) and mixed for 1 minute. Beads werecapture again and supernatant removed. Beads were then washed with 150μl Wash Solution 2 (Ethanol added), captured and supernatant wasremoved. 50 μl of DNase mixture (MagMax turbo DNase Buffer and TurboDNase) was then added to the beads and they were mixed for 10 to 15minutes. After mixing, 100 μl of RNA Rebinding Solution was added andmixed for 3 minutes. Supernatant was removed and magnetic beads werewashed again with 150 μl Wash Solution 2 and mixed for 1 minute andsupernatant was removed completely. The magnetic beads were mixed for 2minutes to dry before RNA was eluted with 50 μl of water.

cDNA Synthesis Using ABI High Capacity cDNA Reverse Transcription Kit(Applied Biosystems, Foster City, Calif., Cat #4368813):

A master mix of 2 μl 10× Buffer, 0.8 μl 125X dNTPs, 2 μl Random primers,1 μl Reverse Transcriptase, 1 μl RNase inhibitor and 3.2 μl of H2O perreaction were added into 10 μl total RNA. cDNA was generated using aBio-Rad C-1000 or S-1000 thermal cycler (Hercules, Calif.) through thefollowing steps: 25° C. 10 min, 37° C. 120 min, 85° C. 5 sec, 4° C.hold.

Real Time PCR:

2 μl of cDNA were added to a master mix containing 0.5 μl GAPDH TaqManProbe (Applied Biosystems Cat #4326317E), 0.5 μl CD274 (PD-L1) TaqManprobe (Applied Biosystems cat #Hs01125301_ml) and 5 μl Roche ProbesMaster Mix (Roche Cat #04887301001) in a total of 10 μl per well in aLightCycler 480 384 well plate (Roche cat #0472974001). Real time PCRwas done in a LightCycler 480 Real Time PCR machine (Roche). Each duplexwas tested in at least two independent transfections. Each transfectionwas assayed by qPCR in duplicate.

Real time data were analyzed using the ΔΔCt method. Each sample wasnormalized to GAPDH expression and knockdown was assessed relative tocells transfected with the non-targeting duplex AD-1955. IC50s weredefined using a 4 parameter fit model in XLfit.

In vitro screening of EGLN1, EGLN2, EGLN3 siRNAs for mRNA suppression

Mouse EGLN1 or EGLN2 or EGLN3 targeting dsRNAs (Tables 2A-F) wereassayed for inhibition of endogenous EGLN1, 2, 3 expression in BNLC12cells, using bDNA (branched DNA) assays to quantify EGLN1,2,3 mRNA.Results from single dose assays were used to select a subset of EGLN1,EGLN2 or EGLN3 dsRNA duplexes for 3 point dose response experiments todetermine relative potency. The most potent siRNA for eachtarget-EGLN1,2,3 was selected for further testing in vivo.

Cell Culture and Transfections:

The mouse liver cell line Bnlc12 (ATCC, Manassas, Va.) were grown tonear confluence at 37° C. in an atmosphere of 5% CO2 in Dulbecco'smodified Eagle's medium (ATCC) supplemented with 10% FBS, streptomycin,and glutamine (ATCC) before being released from the plate bytrypsinization. Reverse transfection was carried out by adding 5 μl ofOpti-MEM to 5 μl of siRNA duplexes per well into a 96-well plate alongwith 10 μl of Opti-MEM plus 0.2 μl of Lipofectamine RNAiMax per well(Invitrogen, Carlsbad Calif. cat #13778-150) and incubated at roomtemperature for 15 minutes. 80 μl of complete growth media withoutantibiotics containing 2×104 Bnlc12 cells were then added. Cells wereincubated for 24 hours prior to preparation of cell lysates for branchedDNA. Single dose experiments were performed at 1 nM final duplexconcentration and dose response experiments were done with 1, 0.1, and0.01 nM.

Branched DNA (bDNA) Assays-QuantiGene 2.0 (Panomics Cat #: QS0011): Usedto Screen Duplexes

After a 24 hour incubation at the dose or doses stated, media wasremoved and cells were lysed in 100 ul Lysis buffer (Epicentertechnologies and 10 μl of Proteinase-K/ml for a final concentration of20 mg/ml) then incubated at 65° C. for 1 hour. 60 μl Working Probe Set(EGLN1, EGLN2 or EGLN3 probe for gene target and GAPDH for endogenouscontrol) and 40 μl of cell-lysate were then added to the Capture Plates.Capture Plates were incubated at 55° C.±1° C. (approx. 16-20 hrs). Thenext day, the Capture Plates were washed 3 times with 1× Wash Buffer(nuclease-free water, Buffer Component 1 and Wash Buffer Component 2),then dried by centrifuging for 1 minute at 240 g. 100 μl ofpre-Amplifier Working Reagent was added to the Capture Plates, whichwere sealed with aluminum foil and incubated for 1 hour at 55° C.±1° C.Following a 1 hour incubation, the wash step was repeated, then 100 μlAmplifier Working Reagent was added. After 1 hour, the wash and drysteps were repeated, and 100 μl Label Probe was added. Capture plateswere incubated 50° C.±1° C. for 1 hour. The plates were then washed with1× Wash Buffer and dried, and then 100 μl Substrate was added to theCapture Plates. Capture Plates were read using the SpectraMaxLuminometer (Molecular Devices, Sunnyvale, Calif.) following 5 to 15minutes incubation. bDNA data were analyzed by (i) subtracting theaverage background (no lysate control) from each triplicate sample, (ii)averaging the resultant triplicate GAPDH (control probe) and EGLN1 orEGLN2 or EGLN3 (experimental probe) values, and then (iii) taking theratio: (experimental probe-background)/(control probe-background).

Results

A summary of the single dose and 3 point dose response curve results forEGLN1, EGLN2, ELGN3-dsRNAs (siRNAs) are presented below in FIGS. 1 and2. Single dose results are expressed as a ratio of EGLN1, or EGLN2, orEGLN3 to GAPDH mRNA in relative light units. The 3 point dose responsedata is expressed as % EGLN1, EGLN2 or EGLN3 mRNA relative to controluntreated, assayed in BnlC12 cells.

Example 3 In Vivo Knock Down of EGLN Genes

In order to determine whether the iRNA agents to the EGLN genes werespecific, knockdown studies were performed using the iRNA agents set outin Table 3.

One siRNA targeting each gene EGLN1 (AD-40894), EGLN2 (AD-40773) andEGLN3 (AD-40758) as well as a mix of all three siRNAs(AD-40894/AD-40773/AD-40758) were formulated in LNP11 (MC3) formulationsto test the ability to knockdown their respective mRNAs in the liver.The experimental outline is below in Table 3 and includes control PBSgroup as well as a control group with an LNP11 formulation containingthe Luciferase siRNA AD-1955. The individual formulations were dosedintravenously at 0.3 mg/kg into female C57B6 mice whereas thecombination mix formulation was dosed at 1 mg/kg.

At 72 hours after dosing the animals were sacrificed. Plasma sampleswere taken and livers were removed, flash frozen then ground intopowder. Small amounts (˜20 mg) of liver powder was disrupted in lysisbuffer for mRNA analysis by branched DNA-QuantiGene 2.0 (Panomics cat #:QS0011). The same bDNA assay and probes used for the screening work wasused. The data is expressed as percent of PBS control ratios of target(EGLN1, 2, 3) mRNA relative to GAPDH mRNA. The results are shown in FIG.3.

It can be seen from FIG. 3 that the iRNA agents for each EGLN gene arespecific to that variant. It is also evident that the mix or cocktailcontaining all three iRNA was effective in reducing the mRNA level ofeach EGLN gene.

TABLE 3 In vivo knockdown of EGLN genes Sample Dose In vitro Group siRNAFormulation Size (n) (mg/kg) IC50 PBS — 4 Luciferase AD-1955 LNP11 5 0.3(control) EGLN1 AD-40894 LNP11 5 0.3 <10 pM EGLN2 AD-40773 LNP11 5 0.3~50 pM EGLN3 AD-40758 LNP11 5 0.3 ~10 pM EGLN1, 2, 3 AD-40894 LNP11 5 1mix (25%) AD-40773 (50%) AD-40758 (25%)

Example 4 In Vivo Induction of Hepatic Erythropoietin (EPO)

In order to determine if knockdown of the three EGLN (HIF prolylhydroxylases) genes simultaneously in the liver will induce downstreamhepatic Epo (Erythropoetin) production, mice were injected IV with iRNAagents directed to each EGLN gene at 0.3 mg/kg or with a mix of allthree EGLN iRNA agents (1 mg/kg) as described in Table 3 above. All iRNAagents were delivered in formulation LNP11. At 72 hours, the animalswere sacrificed and livers taken for bDNA analysis. Serum was also takenfor erythropoietin (EPO) measurements by ELISA kit (R&D Systems)according to the manufacturer's instructions. The results are shown inFIGS. 4A and 4B.

Only the serum samples for the PBS, Luciferase (AD-1955) andLNP11-AD-40894/AD-40773/AD-40758 (EGLN1,2,3 mix) formulation weremeasured for EPO. The data indicate that only serum from animals treatedwith the LNP11-AD-40894/AD-40773/AD-40758 treated animals showed anincrease in EPO levels which was not seen in serum from animals treatedwith PBS or control Luciferase. Therefore, siRNA formulations thatknockdown of all three EGLNs 1, 2, 3 simultaneously in liver can inducean increase in hepatic EPO production measured in serum.

Example 5 In Vivo Dose Response of EGLN in Liver

In order to evaluate the efficacy of the iRNA agents directed to EGLNgenes, dose response studies were conducted for the individual EGLNs inliver. For these studies, mice (3 animals per group) were injected IVwith formulations at doses outlined in Table 4. A mix of EGLN1 and EGLN3formulations were tested to confirm if co-injection of individual LNP11formulations with siRNA against single targets worked as well asinjection of a single formulation with siRNAs against all 3 EGLNtargets. At 72 hours, the animals were sacrificed and livers taken forbDNA and serum taken for Epo measurements by ELISA. The results areshown in FIG. 5.

Results

It was found that all three formulations LNP11-40894, LNP11-40773, andLNP11-40758 dose dependently knocked down the respective mRNA levels ofEGLN1, EGLN2 and EGLN3 after IV administration into C57B6 mice. Therelative IC50 values in vivo were less than 0.033 for LNP11-40894targeting EGLN1, less than 0.033 for LNP11-40773 targeting of EGLN2 andapproximately 0.05 for LNP11-40758. Furthermore, it was possible todetect knockdown of EGLN1 and EGLN3 mRNAs by injection of LNP11-40894and LNP11-40758, suggesting that the siRNAs don't have to be inside thesame liposome together to silence both targets simultaneously.

TABLE 4 In vivo knockdown of EGLN genes Sample Dose In vitro Group siRNAFormulation Size (n) (mg/kg) IC50 PBS — 3 Luciferase AD-1955 LNP11 3 1EGLN1 AD-40894 LNP11 3 (12 1 <10 pM total) 0.33 0.1 0.033 EGLN2 AD-40773LNP11 3 (12 1 ~50 pM total) 0.33 0.1 0.033 EGLN3 AD-40758 LNP11 3 (12 1~10 pM total) 0.33 0.1 0.033 EGLN1, 3 AD-40894 LNP11 3 0.67/ mix (67%)0.33 AD-40758 (33%)

Example 6 In Vivo Production of Erythropoietin and Hematology

In order to determine whether administration of an EGLN iRNA cocktailwas capable of increasing erythropoietin expression in vivo, a study wasdesigned according to Table 5. Female C57B6 mice were dosed IV with PBSor LNP11-1955 luciferase controls or two different mixes of EGLN siRNAformulations at two different doses 1.5 or 1.33 mg/kg respectively. Onday 5 after the first dose plasma samples were taken from each animalfor hematology measurements. On day 7, a second dose of the same amountof a mix of LNP11 formulations or controls was given. Then on day 10 asecond set of plasma samples were taken, animals were sacrificed andlivers were harvested for measurement of EGLN1, EGLN2, EGLN3 and EPOmRNA measurements again by branched DNA analysis. At 72 hours, after the1^(st) dose blood was drawn for hematology measurements including acount of reticulocytes, red blood cells, hemoglobin measurements andhematocrit levels. At 72 hours after the 2^(nd) dose animals weresacrificed and livers taken for bDNA analysis. The Week 1 data are shownin FIGS. 6 and 7 while Week 2 data are shown in FIGS. 8 and 9.

TABLE 5 In vivo knockdown of EGLN genes Sample Dose Group siRNAFormulation size (n) (mg/kg) PBS —  5 Luciferase AD-1955 LNP11  5 1EGLN1, 2, 3 AD-40894 (.375 mpk) LNP11 15 1.5 mix 1 AD-40773 (.75 mpk)AD-40758 (.375 mpk) EGLN1, 2, 3 AD-40894 (.25 mpk) LNP11 10 1.33 mix 2AD-40773 (.5 mpk) AD-40758 (.58 mpk)

It can be seen from FIGS. 6-9 that in both Weeks 1 and 2 that both mix 1and mix 2 result in observable changes. It was found that by day 5 afterthe first dose a large increase in reticulocyte levels and a smallincrease in hematocrit readouts could be detected. By day 10, now after2 injections of the mix of LNP11 formulations with EGLN1, EGLN2 andEGLN3 siRNAs, a considerable increase in reticulocytes versus controlwas observed with an even larger increase in hematocrit, RBC count andhemoglobin levels in the plasma. Collectively, knockdown of EGLN1,2,3led to an increase in liver EPO mRNA and stimulated erythropiesis.

Furthermore, it was found that injection of the mix of 3 LNPs targetingeach EGLN gene resulted in knockdown of all three EGLN targets EGLN1,ELGN2, and EGLN3 while simultaneously leading to an increase of EPO mRNAafter 2 doses at day 10. The data are shown in FIG. 10. The luciferasesiRNA and PBS treated animals had EPO mRNA levels at essentiallybackground levels in the liver whereas in the EGLN siRNA mix treatedgroup there was strong EPO mRNA expression. EGLN1, EGLN2, EGLN3, and EPOmRNA levels were normalized to housekeeping GAPDH control and data isexpressed as a percentage of the PBS control expression.

From these data, it may be concluded that simultaneous knockdown of allthree EGLN genes in the liver is possible with each siRNA in their ownLNP formulations, then mixing them prior to injection. The knockdown ofthe 3 EGLN genes lead to a very dramatic increased expression of EPOmRNA as compared to the PBS control or Luciferase siRNA treated groupswhere liver EPO mRNA was undetectable and at background levels of theassay. Furthermore, it was found that by turning on EPO mRNA expressionin the liver by knocking down the 3 EGLN genes a dramatic increase inerythropoiesis occurs. This could be measured in the blood from dosedanimals where a dramatic increase in reticulocytes or (immature redblood cells) was observed even after the first dose of EGLN1,2,3 siRNAmix treatment. After the second dose it was evident that a significantincrease in not only reticulocytes but also RBC count, hemoglobin andHematocrit measurements was occurring.

Example 7 Design of siRNA Targeting Human EGLN Genes

Oligonucleotide design was carried out to identify siRNAs targeting thegenes encoding the human (Homo sapiens) EGLN 1, 2 and 3 genes. Thedesign process used the EGLN transcript NM_(—)022051.2 for EGLN1 (SEQ IDNO: 390), NM_(—)053046.2 for EGLN2 (SEQ ID NO: 391), and NM_(—)022073.3for EGLN3 (SEQ ID NO: 392). All sequences were obtained from the NCBIRefseq collection. Start refers to the 5′ most position on the target.

It should be understood that while the sequences disclosed in Tables6A-C are represented as 19mer oligonucleotides, the duplexes formed fromsuch oligonucleotides may be 19mer blunt ended constructs or may bemodified by the addition of one or more nucleotides on the 3′ end of thestrands, preferably a dTdT modification to produce 21mer duplexes having2 nucleotide 3′ overhangs.

TABLE 6A Human EGNL1 Single Strands and Duplex Sequences SEQ SEQ ID IDStart Sense Sequence (5′ to 3′) NO. Antisense Sequence (5′ to 3′) NO. 40AGAGACACAAGGCUUUGUU 393 AACAAAGCCUUGUGUCUCU 394 55 UGUUUGCCCCAGAGUAUUA395 UAAUACUCUGGGGCAAACA 396 59 UGCCCCAGAGUAUUAGUUA 397UAACUAAUACUCUGGGGCA 398 60 GCCCCAGAGUAUUAGUUAA 399 UUAACUAAUACUCUGGGGC400 64 CAGAGUAUUAGUUAACCCA 401 UGGGUUAACUAAUACUCUG 402 70AUUAGUUAACCCACCUAGU 403 ACUAGGUGGGUUAACUAAU 404 73 AGUUAACCCACCUAGUGCU405 AGCACUAGGUGGGUUAACU 406 77 AACCCACCUAGUGCUCCUA 407UAGGAGCACUAGGUGGGUU 408 79 CCCACCUAGUGCUCCUAAU 409 AUUAGGAGCACUAGGUGGG410 86 AGUGCUCCUAAUCAUACAA 411 UUGUAUGAUUAGGAGCACU 412 132GCCUCACUCUCUAUUUGUU 413 AACAAAUAGAGAGUGAGGC 414 153 ACCUUCUGUAAAAUUGGUA415 UACCAAUUUUACAGAAGGU 416 168 GGUAGAAUAAUAGUACCCA 417UGGGUACUAUUAUUCUACC 418 170 UAGAAUAAUAGUACCCACU 419 AGUGGGUACUAUUAUUCUA420 171 AGAAUAAUAGUACCCACUU 421 AAGUGGGUACUAUUAUUCU 422 179AGUACCCACUUCAUAGCAU 423 AUGCUAUGAAGUGGGUACU 424 201 AUGAUGAUUAAAUUGGUUA425 UAACCAAUUUAAUCAUCAU 426 235 UUAGAACACAGAUUGGGCA 427UGCCCAAUCUGUGUUCUAA 428 245 GAUUGGGCACAUAACAGCA 429 UGCUGUUAUGUGCCCAAUC430 249 GGGCACAUAACAGCAAGCA 431 UGCUUGCUGUUAUGUGCCC 432 255AUAACAGCAAGCACCACAU 433 AUGUGGUGCUUGCUGUUAU 434 287 AAAUUCCUUUGUGUUGCCU435 AGGCAACACAAAGGAAUUU 436 292 CCUUUGUGUUGCCUUCCGU 437ACGGAAGGCAACACAAAGG 438 293 CUUUGUGUUGCCUUCCGUU 439 AACGGAAGGCAACACAAAG440 295 UUGUGUUGCCUUCCGUUAA 441 UUAACGGAAGGCAACACAA 442 296UGUGUUGCCUUCCGUUAAA 443 UUUAACGGAAGGCAACACA 444 298 UGUUGCCUUCCGUUAAAGU445 ACUUUAACGGAAGGCAACA 446 299 GUUGCCUUCCGUUAAAGUU 447AACUUUAACGGAAGGCAAC 448 336 AAUAAAUACUUGCAUGACA 449 UGUCAUGCAAGUAUUUAUU450 360 AAGUCUCUCUAUAACAUCU 451 AGAUGUUAUAGAGAGACUU 452 368CUAUAACAUCUGAGUAAGU 453 ACUUACUCAGAUGUUAUAG 454 375 AUCUGAGUAAGUGGCGGCU455 AGCCGCCACUUACUCAGAU 456 389 CGGCUGCGACAAUGCUACU 457AGUAGCAUUGUCGCAGCCG 458 394 GCGACAAUGCUACUGGAGU 459 ACUCCAGUAGCAUUGUCGC460 395 CGACAAUGCUACUGGAGUU 461 AACUCCAGUAGCAUUGUCG 462 411GUUCCAGAAUCGUGUUGGU 463 ACCAACACGAUUCUGGAAC 464 428 GUGACAAGAUUGUUCACCA465 UGGUGAACAAUCUUGUCAC 466 434 AGAUUGUUCACCAGCAUAU 467AUAUGCUGGUGAACAAUCU 468 439 GUUCACCAGCAUAUGGUGU 469 ACACCAUAUGCUGGUGAAC470 444 CCAGCAUAUGGUGUGGUGA 471 UCACCACACCAUAUGCUGG 472 453GGUGUGGUGAAAACUCACU 473 AGUGAGUUUUCACCACACC 474 455 UGUGGUGAAAACUCACUAA475 UUAGUGAGUUUUCACCACA 476 457 UGGUGAAAACUCACUAAUU 477AAUUAGUGAGUUUUCACCA 478 458 GGUGAAAACUCACUAAUUU 479 AAAUUAGUGAGUUUUCACC480 488 AGAUUAUUAAGCCUGAAUA 481 UAUUCAGGCUUAAUAAUCU 482 491UUAUUAAGCCUGAAUAGGU 483 ACCUAUUCAGGCUUAAUAA 484 493 AUUAAGCCUGAAUAGGUGA485 UCACCUAUUCAGGCUUAAU 486 494 UUAAGCCUGAAUAGGUGAA 487UUCACCUAUUCAGGCUUAA 488 495 UAAGCCUGAAUAGGUGAAA 489 UUUCACCUAUUCAGGCUUA490 519 GAAAUCAAGGAUCUUUGGA 491 UCCAAAGAUCCUUGAUUUC 492 579UUAAAGUGUUGCAAGUGUU 493 AACACUUGCAACACUUUAA 494 597 UCUAUUUGAUGGAUUAAGU495 ACUUAAUCCAUCAAAUAGA 496 598 CUAUUUGAUGGAUUAAGUA 497UACUUAAUCCAUCAAAUAG 498 599 UAUUUGAUGGAUUAAGUAU 499 AUACUUAAUCCAUCAAAUA500 600 AUUUGAUGGAUUAAGUAUA 501 UAUACUUAAUCCAUCAAAU 502 601UUUGAUGGAUUAAGUAUAU 503 AUAUACUUAAUCCAUCAAA 504 610 UUAAGUAUAUUUAGGAUAU505 AUAUCCUAAAUAUACUUAA 506 611 UAAGUAUAUUUAGGAUAUA 507UAUAUCCUAAAUAUACUUA 508 687 UGAUAUGGACAUCUAUUCU 509 AGAAUAGAUGUCCAUAUCA510 688 GAUAUGGACAUCUAUUCUU 511 AAGAAUAGAUGUCCAUAUC 512 706UUUAAGUAAACUUCAAUGA 513 UCAUUGAAGUUUACUUAAA 514 721 AUGAAAAUAUAUGAGUAGA515 UCUACUCAUAUAUUUUCAU 516 724 AAAAUAUAUGAGUAGAGCA 517UGCUCUACUCAUAUAUUUU 518 725 AAAUAUAUGAGUAGAGCAU 519 AUGCUCUACUCAUAUAUUU520 726 AAUAUAUGAGUAGAGCAUA 521 UAUGCUCUACUCAUAUAUU 522 727AUAUAUGAGUAGAGCAUAU 523 AUAUGCUCUACUCAUAUAU 524 728 UAUAUGAGUAGAGCAUAUA525 UAUAUGCUCUACUCAUAUA 526 730 UAUGAGUAGAGCAUAUAGA 527UCUAUAUGCUCUACUCAUA 528 771 ACCACAGACUGAAAUAGCA 529 UGCUAUUUCAGUCUGUGGU530 827 GGAAUGAGUCCUCCUAGUA 531 UACUAGGAGGACUCAUUCC 532 828GAAUGAGUCCUCCUAGUAA 533 UUACUAGGAGGACUCAUUC 534 829 AAUGAGUCCUCCUAGUAAA535 UUUACUAGGAGGACUCAUU 536 832 GAGUCCUCCUAGUAAAGUU 537AACUUUACUAGGAGGACUC 538 849 UUCCUGCUCUUGUGAAUAA 539 UUAUUCACAAGAGCAGGAA540 859 UGUGAAUAAUUAAGCCUCA 541 UGAGGCUUAAUUAUUCACA 542 868UUAAGCCUCAUGUAUAAUU 543 AAUUAUACAUGAGGCUUAA 544 872 GCCUCAUGUAUAAUUACUA545 UAGUAAUUAUACAUGAGGC 546 901 AAGCCUAAGAAGUAUUAGA 547UCUAAUACUUCUUAGGCUU 548 903 GCCUAAGAAGUAUUAGACU 549 AGUCUAAUACUUCUUAGGC550 973 UUAAAUGCUUAUUUUCGUA 551 UACGAAAAUAAGCAUUUAA 552 978UGCUUAUUUUCGUAAGCCA 553 UGGCUUACGAAAAUAAGCA 554 984 UUUUCGUAAGCCAUGAGAU555 AUCUCAUGGCUUACGAAAA 556 996 AUGAGAUAGCUCCUUUAUA 557UAUAAAGGAGCUAUCUCAU 558 1042 UGGAUUUUAUUAGUGCAAA 559 UUUGCACUAAUAAAAUCCA560 1062 GGCAGAGCUAGCAAUUCCU 561 AGGAAUUGCUAGCUCUGCC 562 1105AUUCAUCCCUCUUUUAGGA 563 UCCUAAAAGAGGGAUGAAU 564 1159 UGCCUCCUGCAUUGGACUA565 UAGUCCAAUGCAGGAGGCA 566 1160 GCCUCCUGCAUUGGACUAU 567AUAGUCCAAUGCAGGAGGC 568 1162 CUCCUGCAUUGGACUAUGU 569 ACAUAGUCCAAUGCAGGAG570 1179 GUGUCUCUGAGUGUAGUAU 571 AUACUACACUCAGAGACAC 572 1185CUGAGUGUAGUAUGACUAA 573 UUAGUCAUACUACACUCAG 574 1186 UGAGUGUAGUAUGACUAAU575 AUUAGUCAUACUACACUCA 576 1187 GAGUGUAGUAUGACUAAUU 577AAUUAGUCAUACUACACUC 578 1189 GUGUAGUAUGACUAAUUCA 579 UGAAUUAGUCAUACUACAC580 1211 GUUUGUCAAGGACUCUCAA 581 UUGAGAGUCCUUGACAAAC 582 1216UCAAGGACUCUCAAUGCAU 583 AUGCAUUGAGAGUCCUUGA 584 1221 GACUCUCAAUGCAUUUGUU585 AACAAAUGCAUUGAGAGUC 586 1233 AUUUGUUGAACAGCCUAAU 587AUUAGGCUGUUCAACAAAU 588 1237 GUUGAACAGCCUAAUUAGU 589 ACUAAUUAGGCUGUUCAAC590 1238 UUGAACAGCCUAAUUAGUA 591 UACUAAUUAGGCUGUUCAA 592 1242ACAGCCUAAUUAGUAAUGU 593 ACAUUACUAAUUAGGCUGU 594 1244 AGCCUAAUUAGUAAUGUCU595 AGACAUUACUAAUUAGGCU 596 1254 GUAAUGUCUGCAACAAUGA 597UCAUUGUUGCAGACAUUAC 598 1285 UUUAAUAAAGCUCUGGGAA 599 UUCCCAGAGCUUUAUUAAA600 1286 UUAAUAAAGCUCUGGGAAA 601 UUUCCCAGAGCUUUAUUAA 602 1293AGCUCUGGGAAAGUAGGAU 603 AUCCUACUUUCCCAGAGCU 604 1296 UCUGGGAAAGUAGGAUACA605 UGUAUCCUACUUUCCCAGA 606 1303 AAGUAGGAUACACAUAAGA 607UCUUAUGUGUAUCCUACUU 608 1308 GGAUACACAUAAGACAGGU 609 ACCUGUCUUAUGUGUAUCC610 1314 ACAUAAGACAGGUCUAGGU 611 ACCUAGACCUGUCUUAUGU 612 1319AGACAGGUCUAGGUCUAAA 613 UUUAGACCUAGACCUGUCU 614 1320 GACAGGUCUAGGUCUAAAU615 AUUUAGACCUAGACCUGUC 616 1323 AGGUCUAGGUCUAAAUUCU 617AGAAUUUAGACCUAGACCU 618 1324 GGUCUAGGUCUAAAUUCUU 619 AAGAAUUUAGACCUAGACC620 1328 UAGGUCUAAAUUCUUUACA 621 UGUAAAGAAUUUAGACCUA 622 1338UUCUUUACAGAAACUUGGA 623 UCCAAGUUUCUGUAAAGAA 624 1403 GUUUCCCAAAGGACAAGCU625 AGCUUGUCCUUUGGGAAAC 626 1434 CAUCCUCUUUCACUUGAUU 627AAUCAAGUGAAAGAGGAUG 628 1470 UUUACGCAUGCAGCAGGAU 629 AUCCUGCUGCAUGCGUAAA630 1471 UUACGCAUGCAGCAGGAUU 631 AAUCCUGCUGCAUGCGUAA 632 1482GCAGGAUUUUAUAACAGUU 633 AACUGUUAUAAAAUCCUGC 634 1572 UGGUUUACAAUAAUUCCUU635 AAGGAAUUAUUGUAAACCA 636 1606 AAUACAUAUUACAACUUUU 637AAAAGUUGUAAUAUGUAUU 638 1625 UAAGUUUGGAAGGCUAUAU 639 AUAUAGCCUUCCAAACUUA640 1626 AAGUUUGGAAGGCUAUAUU 641 AAUAUAGCCUUCCAAACUU 642 1629UUUGGAAGGCUAUAUUUCA 643 UGAAAUAUAGCCUUCCAAA 644 1651 ACUGAAGUUACAGUAUACU645 AGUAUACUGUAACUUCAGU 646 1653 UGAAGUUACAGUAUACUCA 647UGAGUAUACUGUAACUUCA 648 1654 GAAGUUACAGUAUACUCAA 649 UUGAGUAUACUGUAACUUC650 1665 AUACUCAAGUGAUACACAA 651 UUGUGUAUCACUUGAGUAU 652 1673GUGAUACACAAGCCUAGCA 653 UGCUAGGCUUGUGUAUCAC 654 1678 ACACAAGCCUAGCACCCCA655 UGGGGUGCUAGGCUUGUGU 656 1693 CCCACUUUCCACAUAGUGU 657ACACUAUGUGGAAAGUGGG 658 1697 CUUUCCACAUAGUGUUCGA 659 UCGAACACUAUGUGGAAAG660 1698 UUUCCACAUAGUGUUCGAU 661 AUCGAACACUAUGUGGAAA 662 1699UUCCACAUAGUGUUCGAUA 663 UAUCGAACACUAUGUGGAA 664 1700 UCCACAUAGUGUUCGAUAA665 UUAUCGAACACUAUGUGGA 666 1701 CCACAUAGUGUUCGAUAAA 667UUUAUCGAACACUAUGUGG 668 1705 AUAGUGUUCGAUAAAGAUU 669 AAUCUUUAUCGAACACUAU670 1709 UGUUCGAUAAAGAUUGAUA 671 UAUCAAUCUUUAUCGAACA 672 1711UUCGAUAAAGAUUGAUAAA 673 UUUAUCAAUCUUUAUCGAA 674 1721 AUUGAUAAACUCGAAAUCA675 UGAUUUCGAGUUUAUCAAU 676 1723 UGAUAAACUCGAAAUCACA 677UGUGAUUUCGAGUUUAUCA 678 1725 AUAAACUCGAAAUCACAGA 679 UCUGUGAUUUCGAGUUUAU680 1729 ACUCGAAAUCACAGACCUU 681 AAGGUCUGUGAUUUCGAGU 682 1740CAGACCUUUUAAUUCUUAA 683 UUAAGAAUUAAAAGGUCUG 684 1788 GGCUUAUUUCUGGUAAGGU685 ACCUUACCAGAAAUAAGCC 686 1790 CUUAUUUCUGGUAAGGUUU 687AAACCUUACCAGAAAUAAG 688 1829 AAUUGUAUUCAUCCGCGCA 689 UGCGCGGAUGAAUACAAUU690 1832 UGUAUUCAUCCGCGCAGCA 691 UGCUGCGCGGAUGAAUACA 692 1834UAUUCAUCCGCGCAGCACA 693 UGUGCUGCGCGGAUGAAUA 694 1864 AAAUAAAUGUGAGAGUCGU695 ACGACUCUCACAUUUAUUU 696 1866 AUAAAUGUGAGAGUCGUUA 697UAACGACUCUCACAUUUAU 698 1867 UAAAUGUGAGAGUCGUUAA 699 UUAACGACUCUCACAUUUA700 1870 AUGUGAGAGUCGUUAAUGU 701 ACAUUAACGACUCUCACAU 702 1873UGAGAGUCGUUAAUGUAGU 703 ACUACAUUAACGACUCUCA 704 1874 GAGAGUCGUUAAUGUAGUA705 UACUACAUUAACGACUCUC 706 1876 GAGUCGUUAAUGUAGUACU 707AGUACUACAUUAACGACUC 708 1884 AAUGUAGUACUGCUCAUUU 709 AAAUGAGCAGUACUACAUU710 1917 CUUUUCAGGAAUAAUCCCA 711 UGGGAUUAUUCCUGAAAAG 712 1963CAUUGAUUACAUUUAACUU 713 AAGUUAAAUGUAAUCAAUG 714 1966 UGAUUACAUUUAACUUGGU715 ACCAAGUUAAAUGUAAUCA 716 1972 CAUUUAACUUGGUAGCCCA 717UGGGCUACCAAGUUAAAUG 718 1974 UUUAACUUGGUAGCCCAAA 719 UUUGGGCUACCAAGUUAAA720 1978 ACUUGGUAGCCCAAAAUUU 721 AAAUUUUGGGCUACCAAGU 722 1981UGGUAGCCCAAAAUUUCUU 723 AAGAAAUUUUGGGCUACCA 724 1990 AAAAUUUCUUCAUGGGGUU725 AACCCCAUGAAGAAAUUUU 726 2005 GGUUUUGAACUCGGCGGGA 727UCCCGCCGAGUUCAAAACC 728 2006 GUUUUGAACUCGGCGGGAU 729 AUCCCGCCGAGUUCAAAAC730 2007 UUUUGAACUCGGCGGGAUU 731 AAUCCCGCCGAGUUCAAAA 732 2008UUUGAACUCGGCGGGAUUU 733 AAAUCCCGCCGAGUUCAAA 734 2012 AACUCGGCGGGAUUUCAAA735 UUUGAAAUCCCGCCGAGUU 736 2079 UACCUUUAAACUAGGUCGA 737UCGACCUAGUUUAAAGGUA 738 2081 CCUUUAAACUAGGUCGAAA 739 UUUCGACCUAGUUUAAAGG740 2090 UAGGUCGAAACGGGGCGCA 741 UGCGCCCCGUUUCGACCUA 742 2091AGGUCGAAACGGGGCGCAA 743 UUGCGCCCCGUUUCGACCU 744 2093 GUCGAAACGGGGCGCAAGA745 UCUUGCGCCCCGUUUCGAC 746 2097 AAACGGGGCGCAAGAGAUU 747AAUCUCUUGCGCCCCGUUU 748 2102 GGGCGCAAGAGAUUGGAUU 749 AAUCCAAUCUCUUGCGCCC750 2103 GGCGCAAGAGAUUGGAUUA 751 UAAUCCAAUCUCUUGCGCC 752 2104GCGCAAGAGAUUGGAUUAA 753 UUAAUCCAAUCUCUUGCGC 754 2106 GCAAGAGAUUGGAUUAACA755 UGUUAAUCCAAUCUCUUGC 756 2109 AGAGAUUGGAUUAACACCA 757UGGUGUUAAUCCAAUCUCU 758 2113 AUUGGAUUAACACCAUAGU 759 ACUAUGGUGUUAAUCCAAU760 2122 ACACCAUAGUAAUACUUAU 761 AUAAGUAUUACUAUGGUGU 762 2123CACCAUAGUAAUACUUAUU 763 AAUAAGUAUUACUAUGGUG 764 2130 GUAAUACUUAUUUUGUUCU765 AGAACAAAAUAAGUAUUAC 766 2158 CAGGGCUUCUUGAAAUAGA 767UCUAUUUCAAGAAGCCCUG 768 2171 AAUAGAGGCUGUAUGGUGU 769 ACACCAUACAGCCUCUAUU770 2172 AUAGAGGCUGUAUGGUGUA 771 UACACCAUACAGCCUCUAU 772 2179CUGUAUGGUGUAAUGGAAA 773 UUUCCAUUACACCAUACAG 774 2233 UUCAGUCCCAGUUUUGCGU775 ACGCAAAACUGGGACUGAA 776 2235 CAGUCCCAGUUUUGCGUGA 777UCACGCAAAACUGGGACUG 778 2239 CCCAGUUUUGCGUGACCUU 779 AAGGUCACGCAAAACUGGG780 2298 CUGCAAAAUGAGGAUCGCA 781 UGCGAUCCUCAUUUUGCAG 782 2305AUGAGGAUCGCAAUAGCCA 783 UGGCUAUUGCGAUCCUCAU 784 2308 AGGAUCGCAAUAGCCACCU785 AGGUGGCUAUUGCGAUCCU 786 2309 GGAUCGCAAUAGCCACCUU 787AAGGUGGCUAUUGCGAUCC 788 2316 AAUAGCCACCUUGCAACCU 789 AGGUUGCAAGGUGGCUAUU790 2321 CCACCUUGCAACCUUGACU 791 AGUCAAGGUUGCAAGGUGG 792 2328GCAACCUUGACUGGAGCGA 793 UCGCUCCAGUCAAGGUUGC 794 2338 CUGGAGCGAGCCUCGCACA795 UGUGCGAGGCUCGCUCCAG 796 2382 AGCCAUGAUUACGCCGCCU 797AGGCGGCGUAAUCAUGGCU 798 2383 GCCAUGAUUACGCCGCCUU 799 AAGGCGGCGUAAUCAUGGC800 2435 UCCAGCAGGUGUAGGCGCU 801 AGCGCCUACACCUGCUGGA 802 2573AGGGAAAGCGGGCGACCCA 803 UGGGUCGCCCGCUUUCCCU 804 2576 GAAAGCGGGCGACCCACCU805 AGGUGGGUCGCCCGCUUUC 806 2761 GAGCGAGUGGCGCCCGUAU 807AUACGGGCGCCACUCGCUC 808 2766 AGUGGCGCCCGUAUGCCCU 809 AGGGCAUACGGGCGCCACU810 2885 CAGGUUGCCAUUCGCCGCA 811 UGCGGCGAAUGGCAACCUG 812 2887GGUUGCCAUUCGCCGCACA 813 UGUGCGGCGAAUGGCAACC 814 2895 UUCGCCGCACAGGCCCUAU815 AUAGGGCCUGUGCGGCGAA 816 2896 UCGCCGCACAGGCCCUAUU 817AAUAGGGCCUGUGCGGCGA 818 3033 GCGGGUGCAUGGCGCAGUA 819 UACUGCGCCAUGCACCCGC820 3034 CGGGUGCAUGGCGCAGUAA 821 UUACUGCGCCAUGCACCCG 822 3042UGGCGCAGUAACGGCCCCU 823 AGGGGCCGUUACUGCGCCA 824 3043 GGCGCAGUAACGGCCCCUA825 UAGGGGCCGUUACUGCGCC 826 3473 ACGCGGCCAAGGGAAAAGU 827ACUUUUCCCUUGGCCGCGU 828 3608 CCCGCUCAUCGCUGUUCCA 829 UGGAACAGCGAUGAGCGGG830 3626 AGGAGAAGGCGAACCUGUA 831 UACAGGUUCGCCUUCUCCU 832 3650CAAGCAACACGCCCGGGGA 833 UCCCCGGGCGUGUUGCUUG 834 3695 GGCCCAACGGGCAGACGAA835 UUCGUCUGCCCGUUGGGCC 836 3731 AGCUGGCGCUCGAGUACAU 837AUGUACUCGAGCGCCAGCU 838 3734 UGGCGCUCGAGUACAUCGU 839 ACGAUGUACUCGAGCGCCA840 3739 CUCGAGUACAUCGUGCCGU 841 ACGGCACGAUGUACUCGAG 842 3745UACAUCGUGCCGUGCAUGA 843 UCAUGCACGGCACGAUGUA 844 3748 AUCGUGCCGUGCAUGAACA845 UGUUCAUGCACGGCACGAU 846 3752 UGCCGUGCAUGAACAAGCA 847UGCUUGUUCAUGCACGGCA 848 3762 GAACAAGCACGGCAUCUGU 849 ACAGAUGCCGUGCUUGUUC850 3797 UCGGCAAGGAGACCGGACA 851 UGUCCGGUCUCCUUGCCGA 852 3809CCGGACAGCAGAUCGGCGA 853 UCGCCGAUCUGCUGUCCGG 854 3842 UGCACGACACCGGGAAGUU855 AACUUCCCGGUGUCGUGCA 856 3854 GGAAGUUCACGGACGGGCA 857UGCCCGUCCGUGAACUUCC 858 3901 AAGGACAUCCGAGGCGAUA 859 UAUCGCCUCGGAUGUCCUU860 3902 AGGACAUCCGAGGCGAUAA 861 UUAUCGCCUCGGAUGUCCU 862 3904GACAUCCGAGGCGAUAAGA 863 UCUUAUCGCCUCGGAUGUC 864 3905 ACAUCCGAGGCGAUAAGAU865 AUCUUAUCGCCUCGGAUGU 866 3907 AUCCGAGGCGAUAAGAUCA 867UGAUCUUAUCGCCUCGGAU 868 3913 GGCGAUAAGAUCACCUGGA 869 UCCAGGUGAUCUUAUCGCC870 3917 AUAAGAUCACCUGGAUCGA 871 UCGAUCCAGGUGAUCUUAU 872 3922AUCACCUGGAUCGAGGGCA 873 UGCCCUCGAUCCAGGUGAU 874 3939 CAAGGAGCCCGGCUGCGAA875 UUCGCAGCCGGGCUCCUUG 876 3943 GAGCCCGGCUGCGAAACCA 877UGGUUUCGCAGCCGGGCUC 878 3944 AGCCCGGCUGCGAAACCAU 879 AUGGUUUCGCAGCCGGGCU880 3950 GCUGCGAAACCAUUGGGCU 881 AGCCCAAUGGUUUCGCAGC 882 3953GCGAAACCAUUGGGCUGCU 883 AGCAGCCCAAUGGUUUCGC 884 3978 CAGCAUGGACGACCUGAUA885 UAUCAGGUCGUCCAUGCUG 886 3983 UGGACGACCUGAUACGCCA 887UGGCGUAUCAGGUCGUCCA 888 3987 CGACCUGAUACGCCACUGU 889 ACAGUGGCGUAUCAGGUCG890 3988 GACCUGAUACGCCACUGUA 891 UACAGUGGCGUAUCAGGUC 892 3994AUACGCCACUGUAACGGGA 893 UCCCGUUACAGUGGCGUAU 894 4024 UACAAAAUCAAUGGCCGGA895 UCCGGCCAUUGAUUUUGUA 896 4028 AAAUCAAUGGCCGGACGAA 897UUCGUCCGGCCAUUGAUUU 898 4029 AAUCAAUGGCCGGACGAAA 899 UUUCGUCCGGCCAUUGAUU900 4033 AAUGGCCGGACGAAAGCCA 901 UGGCUUUCGUCCGGCCAUU 902 4037GCCGGACGAAAGCCAUGGU 903 ACCAUGGCUUUCGUCCGGC 904 4038 CCGGACGAAAGCCAUGGUU905 AACCAUGGCUUUCGUCCGG 906 4047 AGCCAUGGUUGCUUGUUAU 907AUAACAAGCAACCAUGGCU 908 4054 GUUGCUUGUUAUCCGGGCA 909 UGCCCGGAUAACAAGCAAC910 4055 UUGCUUGUUAUCCGGGCAA 911 UUGCCCGGAUAACAAGCAA 912 4066CCGGGCAAUGGAACGGGUU 913 AACCCGUUCCAUUGCCCGG 914 4067 CGGGCAAUGGAACGGGUUA915 UAACCCGUUCCAUUGCCCG 916 4068 GGGCAAUGGAACGGGUUAU 917AUAACCCGUUCCAUUGCCC 918 4070 GCAAUGGAACGGGUUAUGU 919 ACAUAACCCGUUCCAUUGC920 4076 GAACGGGUUAUGUACGUCA 921 UGACGUACAUAACCCGUUC 922 4077AACGGGUUAUGUACGUCAU 923 AUGACGUACAUAACCCGUU 924 4079 CGGGUUAUGUACGUCAUGU925 ACAUGACGUACAUAACCCG 926 4080 GGGUUAUGUACGUCAUGUU 927AACAUGACGUACAUAACCC 928 4082 GUUAUGUACGUCAUGUUGA 929 UCAACAUGACGUACAUAAC930 4084 UAUGUACGUCAUGUUGAUA 931 UAUCAACAUGACGUACAUA 932 4085AUGUACGUCAUGUUGAUAA 933 UUAUCAACAUGACGUACAU 934 4089 ACGUCAUGUUGAUAAUCCA935 UGGAUUAUCAACAUGACGU 936 4090 CGUCAUGUUGAUAAUCCAA 937UUGGAUUAUCAACAUGACG 938 4113 AGAUGGAAGAUGUGUGACA 939 UGUCACACAUCUUCCAUCU940 4127 UGACAUGUAUAUAUUAUCU 941 AGAUAAUAUAUACAUGUCA 942 4153GACUGGGAUGCCAAGGUAA 943 UUACCUUGGCAUCCCAGUC 944 4163 CCAAGGUAAGUGGAGGUAU945 AUACCUCCACUUACCUUGG 946 4172 GUGGAGGUAUACUUCGAAU 947AUUCGAAGUAUACCUCCAC 948 4173 UGGAGGUAUACUUCGAAUU 949 AAUUCGAAGUAUACCUCCA950 4174 GGAGGUAUACUUCGAAUUU 951 AAAUUCGAAGUAUACCUCC 952 4175GAGGUAUACUUCGAAUUUU 953 AAAAUUCGAAGUAUACCUC 954 4252 UUCUGGUCUGACCGUCGCA955 UGCGACGGUCAGACCAGAA 956 4253 UCUGGUCUGACCGUCGCAA 957UUGCGACGGUCAGACCAGA 958 4257 GUCUGACCGUCGCAACCCU 959 AGGGUUGCGACGGUCAGAC960 4269 CAACCCUCAUGAAGUACAA 961 UUGUACUUCAUGAGGGUUG 962 4294UAUGCUACAAGGUACGCAA 963 UUGCGUACCUUGUAGCAUA 964 4295 AUGCUACAAGGUACGCAAU965 AUUGCGUACCUUGUAGCAU 966 4296 UGCUACAAGGUACGCAAUA 967UAUUGCGUACCUUGUAGCA 968 4297 GCUACAAGGUACGCAAUAA 969 UUAUUGCGUACCUUGUAGC970 4299 UACAAGGUACGCAAUAACU 971 AGUUAUUGCGUACCUUGUA 972 4306UACGCAAUAACUGUUUGGU 973 ACCAAACAGUUAUUGCGUA 974 4307 ACGCAAUAACUGUUUGGUA975 UACCAAACAGUUAUUGCGU 976 4335 AGAUGAGAGAGCACGAGCU 977AGCUCGUGCUCUCUCAUCU 978 4337 AUGAGAGAGCACGAGCUAA 979 UUAGCUCGUGCUCUCUCAU980 4340 AGAGAGCACGAGCUAAAGU 981 ACUUUAGCUCGUGCUCUCU 982 4341GAGAGCACGAGCUAAAGUA 983 UACUUUAGCUCGUGCUCUC 984 4342 AGAGCACGAGCUAAAGUAA985 UUACUUUAGCUCGUGCUCU 986 4356 AGUAAAAUAUCUAACAGGU 987ACCUGUUAGAUAUUUUACU 988 4358 UAAAAUAUCUAACAGGUGA 989 UCACCUGUUAGAUAUUUUA990 4359 AAAAUAUCUAACAGGUGAA 991 UUCACCUGUUAGAUAUUUU 992 4360AAAUAUCUAACAGGUGAAA 993 UUUCACCUGUUAGAUAUUU 994 4379 AAGGUGUGAGGGUUGAACU995 AGUUCAACCCUCACACCUU 996 4381 GGUGUGAGGGUUGAACUCA 997UGAGUUCAACCCUCACACC 998 4384 GUGAGGGUUGAACUCAAUA 999 UAUUGAGUUCAACCCUCAC1000 4386 GAGGGUUGAACUCAAUAAA 1001 UUUAUUGAGUUCAACCCUC 1002 4389GGUUGAACUCAAUAAACCU 1003 AGGUUUAUUGAGUUCAACC 1004 4404ACCUUCAGAUUCGGUCGGU 1005 ACCGACCGAAUCUGAAGGU 1006 4405CCUUCAGAUUCGGUCGGUA 1007 UACCGACCGAAUCUGAAGG 1008 4406CUUCAGAUUCGGUCGGUAA 1009 UUACCGACCGAAUCUGAAG 1010 4407UUCAGAUUCGGUCGGUAAA 1011 UUUACCGACCGAAUCUGAA 1012 4409CAGAUUCGGUCGGUAAAGA 1013 UCUUUACCGACCGAAUCUG 1014 4412AUUCGGUCGGUAAAGACGU 1015 ACGUCUUUACCGACCGAAU 1016 4424AAGACGUCUUCUAGAGCCU 1017 AGGCUCUAGAAGACGUCUU 1018 4425AGACGUCUUCUAGAGCCUU 1019 AAGGCUCUAGAAGACGUCU 1020 4435UAGAGCCUUUGAUCCAGCA 1021 UGCUGGAUCAAAGGCUCUA 1022 4443UUGAUCCAGCAAUACCCCA 1023 UGGGGUAUUGCUGGAUCAA 1024 4451GCAAUACCCCACUUCACCU 1025 AGGUGAAGUGGGGUAUUGC 1026 4461ACUUCACCUACAAUAUUGU 1027 ACAAUAUUGUAGGUGAAGU 1028 4488UGUUAACUUGUGAAUACGA 1029 UCGUAUUCACAAGUUAACA 1030 4489GUUAACUUGUGAAUACGAA 1031 UUCGUAUUCACAAGUUAAC 1032 4494CUUGUGAAUACGAAUAAAU 1033 AUUUAUUCGUAUUCACAAG 1034 4502UACGAAUAAAUGGGAUAAA 1035 UUUAUCCCAUUUAUUCGUA 1036 4525AAUAGACAACCAGUUCGCA 1037 UGCGAACUGGUUGUCUAUU 1038 4526AUAGACAACCAGUUCGCAU 1039 AUGCGAACUGGUUGUCUAU 1040 4527UAGACAACCAGUUCGCAUU 1041 AAUGCGAACUGGUUGUCUA 1042 4528AGACAACCAGUUCGCAUUU 1043 AAAUGCGAACUGGUUGUCU 1044 4608CUUUGUACUGCAUGAUCAA 1045 UUGAUCAUGCAGUACAAAG 1046 4634UCUGUGAUUGCUUACAGGA 1047 UCCUGUAAGCAAUCACAGA 1048 4651GAGGAAGAUAAGCUACUAA 1049 UUAGUAGCUUAUCUUCCUC 1050 4687AUCUGGAUAUGAAAUAAGU 1051 ACUUAUUUCAUAUCCAGAU 1052 4699AAUAAGUGCCCUGUGUAGA 1053 UCUACACAGGGCACUUAUU 1054 4700AUAAGUGCCCUGUGUAGAA 1055 UUCUACACAGGGCACUUAU 1056 4703AGUGCCCUGUGUAGAAUUU 1057 AAAUUCUACACAGGGCACU 1058 4732UAUAUUUUGCCAGAUCUGU 1059 ACAGAUCUGGCAAAAUAUA 1060 4738UUGCCAGAUCUGUUAUCUA 1061 UAGAUAACAGAUCUGGCAA 1062 4741CCAGAUCUGUUAUCUAGCU 1063 AGCUAGAUAACAGAUCUGG 1064 4748UGUUAUCUAGCUGAGUUCA 1065 UGAACUCAGCUAGAUAACA 1066 4749GUUAUCUAGCUGAGUUCAU 1067 AUGAACUCAGCUAGAUAAC 1068 4756AGCUGAGUUCAUUUCAUCU 1069 AGAUGAAAUGAACUCAGCU 1070 4791AAGUUUGAAUUUGGGAUAA 1071 UUAUCCCAAAUUCAAACUU 1072 4812UUUCUAUAUUAGGUACAAU 1073 AUUGUACCUAAUAUAGAAA 1074 4814UCUAUAUUAGGUACAAUUU 1075 AAAUUGUACCUAAUAUAGA 1076 4819AUUAGGUACAAUUUAUCUA 1077 UAGAUAAAUUGUACCUAAU 1078 4820UUAGGUACAAUUUAUCUAA 1079 UUAGAUAAAUUGUACCUAA 1080 4821UAGGUACAAUUUAUCUAAA 1081 UUUAGAUAAAUUGUACCUA 1082 4823GGUACAAUUUAUCUAAACU 1083 AGUUUAGAUAAAUUGUACC 1084 4870CUCAAAAUAACAUCAAUCU 1085 AGAUUGAUGUUAUUUUGAG 1086 4893UUGUAAACCUGUUCAUACU 1087 AGUAUGAACAGGUUUACAA 1088 4894UGUAAACCUGUUCAUACUA 1089 UAGUAUGAACAGGUUUACA 1090 4897AAACCUGUUCAUACUAUUA 1091 UAAUAGUAUGAACAGGUUU 1092 4909ACUAUUAAAUUUUGCCCUA 1093 UAGGGCAAAAUUUAAUAGU 1094 4919UUUGCCCUAAAAGACCUCU 1095 AGAGGUCUUUUAGGGCAAA 1096 4920UUGCCCUAAAAGACCUCUU 1097 AAGAGGUCUUUUAGGGCAA 1098 4929AAGACCUCUUAAUAAUGAU 1099 AUCAUUAUUAAGAGGUCUU 1100 4930AGACCUCUUAAUAAUGAUU 1101 AAUCAUUAUUAAGAGGUCU 1102 4933CCUCUUAAUAAUGAUUGUU 1103 AACAAUCAUUAUUAAGAGG 1104 4952GCCAGUGACUGAUGAUUAA 1105 UUAAUCAUCAGUCACUGGC 1106 4953CCAGUGACUGAUGAUUAAU 1107 AUUAAUCAUCAGUCACUGG 1108 4954CAGUGACUGAUGAUUAAUU 1109 AAUUAAUCAUCAGUCACUG 1110 4997GAGCACUUUAAUUACAACU 1111 AGUUGUAAUUAAAGUGCUC 1112 5031UUUGUAGUCCUUCCUUACA 1113 UGUAAGGAAGGACUACAAA 1114 5035UAGUCCUUCCUUACACUAA 1115 UUAGUGUAAGGAAGGACUA 1116 5048CACUAAUUUGAACUGUUAA 1117 UUAACAGUUCAAAUUAGUG 1118 5084UUGACAUUGUCAAUAACGA 1119 UCGUUAUUGACAAUGUCAA 1120 5085UGACAUUGUCAAUAACGAA 1121 UUCGUUAUUGACAAUGUCA 1122 5086GACAUUGUCAAUAACGAAA 1123 UUUCGUUAUUGACAAUGUC 1124 5089AUUGUCAAUAACGAAACCU 1125 AGGUUUCGUUAUUGACAAU 1126 5090UUGUCAAUAACGAAACCUA 1127 UAGGUUUCGUUAUUGACAA 1128 5091UGUCAAUAACGAAACCUAA 1129 UUAGGUUUCGUUAUUGACA 1130 5095AAUAACGAAACCUAAUUGU 1131 ACAAUUAGGUUUCGUUAUU 1132 5096AUAACGAAACCUAAUUGUA 1133 UACAAUUAGGUUUCGUUAU 1134 5105CCUAAUUGUAAAACAGUCA 1135 UGACUGUUUUACAAUUAGG 1136 5111UGUAAAACAGUCACCAUUU 1137 AAAUGGUGACUGUUUUACA 1138 5120GUCACCAUUUACUACCAAU 1139 AUUGGUAGUAAAUGGUGAC 1140 5121UCACCAUUUACUACCAAUA 1141 UAUUGGUAGUAAAUGGUGA 1142 5122CACCAUUUACUACCAAUAA 1143 UUAUUGGUAGUAAAUGGUG 1144 5124CCAUUUACUACCAAUAACU 1145 AGUUAUUGGUAGUAAAUGG 1146 5399CCUAGGCUGGGGUUUAAGU 1147 ACUUAAACCCCAGCCUAGG 1148 5404GCUGGGGUUUAAGUUAAAU 1149 AUUUAACUUAAACCCCAGC 1150 5405CUGGGGUUUAAGUUAAAUU 1151 AAUUUAACUUAAACCCCAG 1152 5432AACUAAAGUGACUGGCACU 1153 AGUGCCAGUCACUUUAGUU 1154 5474GCUUCAAGUUCCUAAGAUA 1155 UAUCUUAGGAACUUGAAGC 1156 5481GUUCCUAAGAUAAGGGCUU 1157 AAGCCCUUAUCUUAGGAAC 1158 5484CCUAAGAUAAGGGCUUUCU 1159 AGAAAGCCCUUAUCUUAGG 1160 5511CAGGUGUAUGUAUCCUCUA 1161 UAGAGGAUACAUACACCUG 1162 5513GGUGUAUGUAUCCUCUAGA 1163 UCUAGAGGAUACAUACACC 1164 5517UAUGUAUCCUCUAGAUGUA 1165 UACAUCUAGAGGAUACAUA 1166 5523UCCUCUAGAUGUAGACAAU 1167 AUUGUCUACAUCUAGAGGA 1168 5524CCUCUAGAUGUAGACAAUA 1169 UAUUGUCUACAUCUAGAGG 1170 5543AUGUCCCAUUUCUAAGUCU 1171 AGACUUAGAAAUGGGACAU 1172 5544UGUCCCAUUUCUAAGUCUU 1173 AAGACUUAGAAAUGGGACA 1174 5574UCUCCUUAAAUUGAUUGUA 1175 UACAAUCAAUUUAAGGAGA 1176 5580UAAAUUGAUUGUACUUCCA 1177 UGGAAGUACAAUCAAUUUA 1178 5581AAAUUGAUUGUACUUCCAA 1179 UUGGAAGUACAAUCAAUUU 1180 5624AUACUGUGAUCUAUCUGAU 1181 AUCAGAUAGAUCACAGUAU 1182 5659UGUCUCUGUUGAAGAGCAU 1183 AUGCUCUUCAACAGAGACA 1184 5662CUCUGUUGAAGAGCAUCAA 1185 UUGAUGCUCUUCAACAGAG 1186 5673AGCAUCAAGGGGAGAUUAU 1187 AUAAUCUCCCCUUGAUGCU 1188 5676AUCAAGGGGAGAUUAUGUA 1189 UACAUAAUCUCCCCUUGAU 1190 5678CAAGGGGAGAUUAUGUACA 1191 UGUACAUAAUCUCCCCUUG 1192 5711UGUGGUGUUACUGACGGAA 1193 UUCCGUCAGUAACACCACA 1194 5714GGUGUUACUGACGGAAUGU 1195 ACAUUCCGUCAGUAACACC 1196 5717GUUACUGACGGAAUGUGCA 1197 UGCACAUUCCGUCAGUAAC 1198 5723GACGGAAUGUGCAGUAACU 1199 AGUUACUGCACAUUCCGUC 1200 5738AACUCCUCAGAUAUCUGUU 1201 AACAGAUAUCUGAGGAGUU 1202 5740CUCCUCAGAUAUCUGUUAA 1203 UUAACAGAUAUCUGAGGAG 1204 5782GCCUUCUUACCUGUACUGA 1205 UCAGUACAGGUAAGAAGGC 1206 5792CUGUACUGAAAGAUGCUUA 1207 UAAGCAUCUUUCAGUACAG 1208 5795UACUGAAAGAUGCUUAGCU 1209 AGCUAAGCAUCUUUCAGUA 1210 5799GAAAGAUGCUUAGCUUAGA 1211 UCUAAGCUAAGCAUCUUUC 1212 5801AAGAUGCUUAGCUUAGAAA 1213 UUUCUAAGCUAAGCAUCUU 1214 5860UCAUGGGUUUUCUUAUUUA 1215 UAAAUAAGAAAACCCAUGA 1216 5915AAGGCCUCACAUACAUGUU 1217 AACAUGUAUGUGAGGCCUU 1218 5917GGCCUCACAUACAUGUUAU 1219 AUAACAUGUAUGUGAGGCC 1220 5918GCCUCACAUACAUGUUAUU 1221 AAUAACAUGUAUGUGAGGC 1222 5944UGAAUUGGGACGGAUGUCU 1223 AGACAUCCGUCCCAAUUCA 1224 5945GAAUUGGGACGGAUGUCUU 1225 AAGACAUCCGUCCCAAUUC 1226 5948UUGGGACGGAUGUCUUAGA 1227 UCUAAGACAUCCGUCCCAA 1228 5950GGGACGGAUGUCUUAGACU 1229 AGUCUAAGACAUCCGUCCC 1230 5961CUUAGACUUCACUUUCCUA 1231 UAGGAAAGUGAAGUCUAAG 1232 5965GACUUCACUUUCCUAGGCU 1233 AGCCUAGGAAAGUGAAGUC 1234 5966ACUUCACUUUCCUAGGCUU 1235 AAGCCUAGGAAAGUGAAGU 1236 5967CUUCACUUUCCUAGGCUUU 1237 AAAGCCUAGGAAAGUGAAG 1238 5968UUCACUUUCCUAGGCUUUU 1239 AAAAGCCUAGGAAAGUGAA 1240 5994ACCUAAAGGGUGGUAUCCA 1241 UGGAUACCACCCUUUAGGU 1242 5997UAAAGGGUGGUAUCCAUAU 1243 AUAUGGAUACCACCCUUUA 1244 5998AAAGGGUGGUAUCCAUAUU 1245 AAUAUGGAUACCACCCUUU 1246 6004UGGUAUCCAUAUUUUGCGU 1247 ACGCAAAAUAUGGAUACCA 1248 6006GUAUCCAUAUUUUGCGUGA 1249 UCACGCAAAAUAUGGAUAC 1250 6007UAUCCAUAUUUUGCGUGAA 1251 UUCACGCAAAAUAUGGAUA 1252 6008AUCCAUAUUUUGCGUGAAU 1253 AUUCACGCAAAAUAUGGAU 1254 6010CCAUAUUUUGCGUGAAUUA 1255 UAAUUCACGCAAAAUAUGG 1256 6017UUGCGUGAAUUAUGGGUGU 1257 ACACCCAUAAUUCACGCAA 1258 6024AAUUAUGGGUGUAAGACCU 1259 AGGUCUUACACCCAUAAUU 1260 6025AUUAUGGGUGUAAGACCUU 1261 AAGGUCUUACACCCAUAAU 1262 6038GACCUUGCCCACUUAGGUU 1263 AACCUAAGUGGGCAAGGUC 1264 6048ACUUAGGUUUUCUAUCUCU 1265 AGAGAUAGAAAACCUAAGU 1266 6050UUAGGUUUUCUAUCUCUGU 1267 ACAGAGAUAGAAAACCUAA 1268 6057UUCUAUCUCUGUCCUUGAU 1269 AUCAAGGACAGAGAUAGAA 1270 6059CUAUCUCUGUCCUUGAUCU 1271 AGAUCAAGGACAGAGAUAG 1272 6083GCCAAAAUGUGAGUAUACA 1273 UGUAUACUCACAUUUUGGC 1274 6085CAAAAUGUGAGUAUACAGA 1275 UCUGUAUACUCACAUUUUG 1276 6137AGCAUCUGUAUAGUUUGUA 1277 UACAAACUAUACAGAUGCU 1278 6153GUAUUCAAUUUGAGACCUU 1279 AAGGUCUCAAAUUGAAUAC 1280 6167ACCUUUUCUAUGGGAAGCU 1281 AGCUUCCCAUAGAAAAGGU 1282 6169CUUUUCUAUGGGAAGCUCA 1283 UGAGCUUCCCAUAGAAAAG 1284 6206UUGCCAUUGCUAUUCAUGU 1285 ACAUGAAUAGCAAUGGCAA 1286 6273GGGAUUGAAUGUUCAGUAU 1287 AUACUGAACAUUCAAUCCC 1288 6290AUAGUGAUCUCACUUAGGA 1289 UCCUAAGUGAGAUCACUAU 1290 6318GGAGAAAGUGAUAGUUUAU 1291 AUAAACUAUCACUUUCUCC 1292 6341UUUUCCUCGCCCAUAUUCA 1293 UGAAUAUGGGCGAGGAAAA 1294 6344UCCUCGCCCAUAUUCAGUU 1295 AACUGAAUAUGGGCGAGGA 1296 6345CCUCGCCCAUAUUCAGUUU 1297 AAACUGAAUAUGGGCGAGG 1298 6346CUCGCCCAUAUUCAGUUUU 1299 AAAACUGAAUAUGGGCGAG 1300 6348CGCCCAUAUUCAGUUUUGU 1301 ACAAAACUGAAUAUGGGCG 1302 6389AGAUGAUAACAUCACAUCU 1303 AGAUGUGAUGUUAUCAUCU 1304 6400UCACAUCUCUACAGUAAGU 1305 ACUUACUGUAGAGAUGUGA 1306 6431CCAACCCAGGAGCGCAAGU 1307 ACUUGCGCUCCUGGGUUGG 1308 6432CAACCCAGGAGCGCAAGUU 1309 AACUUGCGCUCCUGGGUUG 1310 6458CCAUCUGGUCUAUAGUACA 1311 UGUACUAUAGACCAGAUGG 1312 6469AUAGUACAGUGCGCGGCGU 1313 ACGCCGCGCACUGUACUAU 1314 6470UAGUACAGUGCGCGGCGUU 1315 AACGCCGCGCACUGUACUA 1316 6471AGUACAGUGCGCGGCGUUA 1317 UAACGCCGCGCACUGUACU 1318 6476AGUGCGCGGCGUUAGGCCA 1319 UGGCCUAACGCCGCGCACU 1320 6478UGCGCGGCGUUAGGCCACA 1321 UGUGGCCUAACGCCGCGCA 1322 6479GCGCGGCGUUAGGCCACAA 1323 UUGUGGCCUAACGCCGCGC 1324 6484GCGUUAGGCCACAACUCAA 1325 UUGAGUUGUGGCCUAACGC 1326 6485CGUUAGGCCACAACUCAAA 1327 UUUGAGUUGUGGCCUAACG 1328 6516UUUAGGGUUAGUAGAAAUU 1329 AAUUUCUACUAACCCUAAA 1330 6537UUUAUGUUGAUGGGAGGUU 1331 AACCUCCCAUCAACAUAAA 1332 6548GGGAGGUUUGUUUGAUUGU 1333 ACAAUCAAACAAACCUCCC 1334 6581ACAGCCUUUUAAUUUGGGA 1335 UCCCAAAUUAAAAGGCUGU 1336 6599AGCCCCUGUUGUCAUUCAA 1337 UUGAAUGACAACAGGGGCU 1338 6609GUCAUUCAAAUGUGUACCU 1339 AGGUACACAUUUGAAUGAC 1340 6612AUUCAAAUGUGUACCUCUA 1341 UAGAGGUACACAUUUGAAU 1342 6656CUAUCUGUGGGUUGUGCUU 1343 AAGCACAACCCACAGAUAG 1344 6669GUGCUUGCCAGACAGGUCU 1345 AGACCUGUCUGGCAAGCAC 1346 6716UAUACUCUCUUAGGAAUCA 1347 UGAUUCCUAAGAGAGUAUA 1348 6747CAAGAAAUCAGGAUGGCCA 1349 UGGCCAUCCUGAUUUCUUG 1350 6788CAUGUUAGUGGGACUAUUA 1351 UAAUAGUCCCACUAACAUG 1352 6800ACUAUUAACUUGUCACCAA 1353 UUGGUGACAAGUUAAUAGU 1354 6862AUAUGUGUUUAAUCCUGGU 1355 ACCAGGAUUAAACACAUAU 1356 6868GUUUAAUCCUGGUUAAAGA 1357 UCUUUAACCAGGAUUAAAC 1358 6869UUUAAUCCUGGUUAAAGAU 1359 AUCUUUAACCAGGAUUAAA 1360 6911UUCAACACAUUAACCAGCU 1361 AGCUGGUUAAUGUGUUGAA 1362 6942CCUUUAUCAAGAGUAGGCA 1363 UGCCUACUCUUGAUAAAGG 1364 6943CUUUAUCAAGAGUAGGCAA 1365 UUGCCUACUCUUGAUAAAG 1366 6974UUCAUAUACAGAUAGACUA 1367 UAGUCUAUCUGUAUAUGAA 1368 6985AUAGACUAUAAAGUCAUGU 1369 ACAUGACUUUAUAGUCUAU 1370 6986UAGACUAUAAAGUCAUGUA 1371 UACAUGACUUUAUAGUCUA 1372 7040CAAGUUGCUUGUAAAGCUA 1373 UAGCUUUACAAGCAACUUG 1374 7041AAGUUGCUUGUAAAGCUAA 1375 UUAGCUUUACAAGCAACUU 1376 7045UGCUUGUAAAGCUAAUCUA 1377 UAGAUUAGCUUUACAAGCA 1378 7046GCUUGUAAAGCUAAUCUAA 1379 UUAGAUUAGCUUUACAAGC 1380

TABLE 6B Human EGNL2 Single Strands and Duplex Sequences SEQ SEQ ID IDStart Sense Sequence (5′ to 3′) NO. Antisense Sequence (5′ to 3′) NO. 64CCACCCUGAAGGGUCCCUU 1381 AAGGGACCCUUCAGGGUGG 1382 76 GUCCCUUCCCAAGCCCUUA1383 UAAGGGCUUGGGAAGGGAC 1384 80 CUUCCCAAGCCCUUAGGGA 1385UCCCUAAGGGCUUGGGAAG 1386 85 CAAGCCCUUAGGGACCGCA 1387 UGCGGUCCCUAAGGGCUUG1388 93 UAGGGACCGCAGAGGACUU 1389 AAGUCCUCUGCGGUCCCUA 1390 98ACCGCAGAGGACUUGGGGA 1391 UCCCCAAGUCCUCUGCGGU 1392 108ACUUGGGGACCAGCAAGCA 1393 UGCUUGCUGGUCCCCAAGU 1394 109CUUGGGGACCAGCAAGCAA 1395 UUGCUUGCUGGUCCCCAAG 1396 115GACCAGCAAGCAACCCCCA 1397 UGGGGGUUGCUUGCUGGUC 1398 125CAACCCCCAGGGCACGAGA 1399 UCUCGUGCCCUGGGGGUUG 1400 126AACCCCCAGGGCACGAGAA 1401 UUCUCGUGCCCUGGGGGUU 1402 128CCCCCAGGGCACGAGAAGA 1403 UCUUCUCGUGCCCUGGGGG 1404 137CACGAGAAGAGCUCUUGCU 1405 AGCAAGAGCUCUUCUCGUG 1406 139CGAGAAGAGCUCUUGCUGU 1407 ACAGCAAGAGCUCUUCUCG 1408 141AGAAGAGCUCUUGCUGUCU 1409 AGACAGCAAGAGCUCUUCU 1410 195GCCCCCAGCUGCAUCAAGU 1411 ACUUGAUGCAGCUGGGGGC 1412 244CACCAUGGGCCCGGGCGGU 1413 ACCGCCCGGGCCCAUGGUG 1414 253CCCGGGCGGUGCCCUCCAU 1415 AUGGAGGGCACCGCCCGGG 1416 266CUCCAUGCCCGGGGGAUGA 1417 UCAUCCCCCGGGCAUGGAG 1418 269CAUGCCCGGGGGAUGAAGA 1419 UCUUCAUCCCCCGGGCAUG 1420 271UGCCCGGGGGAUGAAGACA 1421 UGUCUUCAUCCCCCGGGCA 1422 273CCCGGGGGAUGAAGACACU 1423 AGUGUCUUCAUCCCCCGGG 1424 276GGGGGAUGAAGACACUGCU 1425 AGCAGUGUCUUCAUCCCCC 1426 310UGCCAGCCGCAGCCCCUAA 1427 UUAGGGGCUGCGGCUGGCA 1428 314AGCCGCAGCCCCUAAGUCA 1429 UGACUUAGGGGCUGCGGCU 1430 318GCAGCCCCUAAGUCAGGCU 1431 AGCCUGACUUAGGGGCUGC 1432 320AGCCCCUAAGUCAGGCUCU 1433 AGAGCCUGACUUAGGGGCU 1434 324CCUAAGUCAGGCUCUCCCU 1435 AGGGAGAGCCUGACUUAGG 1436 328AGUCAGGCUCUCCCUCAGU 1437 ACUGAGGGAGAGCCUGACU 1438 329GUCAGGCUCUCCCUCAGUU 1439 AACUGAGGGAGAGCCUGAC 1440 340CCUCAGUUACCAGGGUCUU 1441 AAGACCCUGGUAACUGAGG 1442 343CAGUUACCAGGGUCUUCGU 1443 ACGAAGACCCUGGUAACUG 1444 345GUUACCAGGGUCUUCGUCA 1445 UGACGAAGACCCUGGUAAC 1446 347UACCAGGGUCUUCGUCAGA 1447 UCUGACGAAGACCCUGGUA 1448 398UGGGAGUGGAGAGUUACCU 1449 AGGUAACUCUCCACUCCCA 1450 441CCACUGUCCAGGAGUGCCU 1451 AGGCACUCCUGGACAGUGG 1452 456GCCUAGUGAGGCCUCGGCA 1453 UGCCGAGGCCUCACUAGGC 1454 516CAGCCCUCUUCGGGACGGU 1455 ACCGUCCCGAAGAGGGCUG 1456 518GCCCUCUUCGGGACGGUUU 1457 AAACCGUCCCGAAGAGGGC 1458 519CCCUCUUCGGGACGGUUUU 1459 AAAACCGUCCCGAAGAGGG 1460 527GGGACGGUUUUGGCGGGCA 1461 UGCCCGCCAAAACCGUCCC 1462 531CGGUUUUGGCGGGCAGGAU 1463 AUCCUGCCCGCCAAAACCG 1464 534UUUUGGCGGGCAGGAUGGU 1465 ACCAUCCUGCCCGCCAAAA 1466 561GCGGCCGCUGCAGAGUGAA 1467 UUCACUCUGCAGCGGCCGC 1468 567GCUGCAGAGUGAAGGCGCU 1469 AGCGCCUUCACUCUGCAGC 1470 583GCUGCAGCGCUGGUCACCA 1471 UGGUGACCAGCGCUGCAGC 1472 593UGGUCACCAAGGGGUGCCA 1473 UGGCACCCCUUGGUGACCA 1474 598ACCAAGGGGUGCCAGCGAU 1475 AUCGCUGGCACCCCUUGGU 1476 599CCAAGGGGUGCCAGCGAUU 1477 AAUCGCUGGCACCCCUUGG 1478 603GGGGUGCCAGCGAUUGGCA 1479 UGCCAAUCGCUGGCACCCC 1480 615AUUGGCAGCCCAGGGCGCA 1481 UGCGCCCUGGGCUGCCAAU 1482 637CCUGAGGCCCCCAAACGGA 1483 UCCGUUUGGGGGCCUCAGG 1484 638CUGAGGCCCCCAAACGGAA 1485 UUCCGUUUGGGGGCCUCAG 1486 639UGAGGCCCCCAAACGGAAA 1487 UUUCCGUUUGGGGGCCUCA 1488 640GAGGCCCCCAAACGGAAAU 1489 AUUUCCGUUUGGGGGCCUC 1490 650AACGGAAAUGGGCCGAGGA 1491 UCCUCGGCCCAUUUCCGUU 1492 651ACGGAAAUGGGCCGAGGAU 1493 AUCCUCGGCCCAUUUCCGU 1494 654GAAAUGGGCCGAGGAUGGU 1495 ACCAUCCUCGGCCCAUUUC 1496 685UCACCCAGCAAACGGCCCU 1497 AGGGCCGUUUGCUGGGUGA 1498 704GGGCCAGGCAAGAGAACCA 1499 UGGUUCUCUUGCCUGGCCC 1500 803CGCUGCCCUCUGCGCCCGA 1501 UCGGGCGCAGAGGGCAGCG 1502 824GCCUGGCCCUGGACUAUAU 1503 AUAUAGUCCAGGGCCAGGC 1504 827UGGCCCUGGACUAUAUCGU 1505 ACGAUAUAGUCCAGGGCCA 1506 835GACUAUAUCGUGCCCUGCA 1507 UGCAGGGCACGAUAUAGUC 1508 836ACUAUAUCGUGCCCUGCAU 1509 AUGCAGGGCACGAUAUAGU 1510 842UCGUGCCCUGCAUGCGGUA 1511 UACCGCAUGCAGGGCACGA 1512 844GUGCCCUGCAUGCGGUACU 1513 AGUACCGCAUGCAGGGCAC 1514 845UGCCCUGCAUGCGGUACUA 1515 UAGUACCGCAUGCAGGGCA 1516 851GCAUGCGGUACUACGGCAU 1517 AUGCCGUAGUACCGCAUGC 1518 853AUGCGGUACUACGGCAUCU 1519 AGAUGCCGUAGUACCGCAU 1520 857GGUACUACGGCAUCUGCGU 1521 ACGCAGAUGCCGUAGUACC 1522 859UACUACGGCAUCUGCGUCA 1523 UGACGCAGAUGCCGUAGUA 1524 863ACGGCAUCUGCGUCAAGGA 1525 UCCUUGACGCAGAUGCCGU 1526 868AUCUGCGUCAAGGACAGCU 1527 AGCUGUCCUUGACGCAGAU 1528 896CAGCACUGGGCGGUCGCGU 1529 ACGCGACCGCCCAGUGCUG 1530 899CACUGGGCGGUCGCGUGCU 1531 AGCACGCGACCGCCCAGUG 1532 927GGAGGCCCUCAAACGGGGU 1533 ACCCCGUUUGAGGGCCUCC 1534 935UCAAACGGGGUGGGCGCCU 1535 AGGCGCCCACCCCGUUUGA 1536 939ACGGGGUGGGCGCCUGCGA 1537 UCGCAGGCGCCCACCCCGU 1538 947GGCGCCUGCGAGACGGGCA 1539 UGCCCGUCUCGCAGGCGCC 1540 967CUAGUGAGCCAGAGGGCGA 1541 UCGCCCUCUGGCUCACUAG 1542 968UAGUGAGCCAGAGGGCGAU 1543 AUCGCCCUCUGGCUCACUA 1544 982GCGAUCCCGCCGCGCAGCA 1545 UGCUGCGCGGCGGGAUCGC 1546 983CGAUCCCGCCGCGCAGCAU 1547 AUGCUGCGCGGCGGGAUCG 1548 987CCCGCCGCGCAGCAUCCGU 1549 ACGGAUGCUGCGCGGCGGG 1550 992CGCGCAGCAUCCGUGGGGA 1551 UCCCCACGGAUGCUGCGCG 1552 999CAUCCGUGGGGACCAGAUU 1553 AAUCUGGUCCCCACGGAUG 1554 1011CCAGAUUGCCUGGGUGGAA 1555 UUCCACCCAGGCAAUCUGG 1556 1019CCUGGGUGGAAGGCCAUGA 1557 UCAUGGCCUUCCACCCAGG 1558 1020CUGGGUGGAAGGCCAUGAA 1559 UUCAUGGCCUUCCACCCAG 1560 1032CCAUGAACCAGGCUGUCGA 1561 UCGACAGCCUGGUUCAUGG 1562 1033CAUGAACCAGGCUGUCGAA 1563 UUCGACAGCCUGGUUCAUG 1564 1036GAACCAGGCUGUCGAAGCA 1565 UGCUUCGACAGCCUGGUUC 1566 1041AGGCUGUCGAAGCAUUGGU 1567 ACCAAUGCUUCGACAGCCU 1568 1046GUCGAAGCAUUGGUGCCCU 1569 AGGGCACCAAUGCUUCGAC 1570 1048CGAAGCAUUGGUGCCCUCA 1571 UGAGGGCACCAAUGCUUCG 1572 1049GAAGCAUUGGUGCCCUCAU 1573 AUGAGGGCACCAAUGCUUC 1574 1058GUGCCCUCAUGGCCCAUGU 1575 ACAUGGGCCAUGAGGGCAC 1576 1070CCCAUGUGGACGCCGUCAU 1577 AUGACGGCGUCCACAUGGG 1578 1076UGGACGCCGUCAUCCGCCA 1579 UGGCGGAUGACGGCGUCCA 1580 1078GACGCCGUCAUCCGCCACU 1581 AGUGGCGGAUGACGGCGUC 1582 1100CAGGGCGGCUGGGCAGCUA 1583 UAGCUGCCCAGCCGCCCUG 1584 1103GGCGGCUGGGCAGCUAUGU 1585 ACAUAGCUGCCCAGCCGCC 1586 1106GGCUGGGCAGCUAUGUCAU 1587 AUGACAUAGCUGCCCAGCC 1588 1117UAUGUCAUCAACGGGCGCA 1589 UGCGCCCGUUGAUGACAUA 1590 1120GUCAUCAACGGGCGCACCA 1591 UGGUGCGCCCGUUGAUGAC 1592 1121UCAUCAACGGGCGCACCAA 1593 UUGGUGCGCCCGUUGAUGA 1594 1126AACGGGCGCACCAAGGCCA 1595 UGGCCUUGGUGCGCCCGUU 1596 1137CAAGGCCAUGGUGGCGUGU 1597 ACACGCCACCAUGGCCUUG 1598 1143CAUGGUGGCGUGUUACCCA 1599 UGGGUAACACGCCACCAUG 1600 1148UGGCGUGUUACCCAGGCAA 1601 UUGCCUGGGUAACACGCCA 1602 1154GUUACCCAGGCAACGGGCU 1603 AGCCCGUUGCCUGGGUAAC 1604 1159CCAGGCAACGGGCUCGGGU 1605 ACCCGAGCCCGUUGCCUGG 1606 1160CAGGCAACGGGCUCGGGUA 1607 UACCCGAGCCCGUUGCCUG 1608 1163GCAACGGGCUCGGGUACGU 1609 ACGUACCCGAGCCCGUUGC 1610 1164CAACGGGCUCGGGUACGUA 1611 UACGUACCCGAGCCCGUUG 1612 1165AACGGGCUCGGGUACGUAA 1613 UUACGUACCCGAGCCCGUU 1614 1169GGCUCGGGUACGUAAGGCA 1615 UGCCUUACGUACCCGAGCC 1616 1172UCGGGUACGUAAGGCACGU 1617 ACGUGCCUUACGUACCCGA 1618 1173CGGGUACGUAAGGCACGUU 1619 AACGUGCCUUACGUACCCG 1620 1175GGUACGUAAGGCACGUUGA 1621 UCAACGUGCCUUACGUACC 1622 1177UACGUAAGGCACGUUGACA 1623 UGUCAACGUGCCUUACGUA 1624 1178ACGUAAGGCACGUUGACAA 1625 UUGUCAACGUGCCUUACGU 1626 1179CGUAAGGCACGUUGACAAU 1627 AUUGUCAACGUGCCUUACG 1628 1190UUGACAAUCCCCACGGCGA 1629 UCGCCGUGGGGAUUGUCAA 1630 1191UGACAAUCCCCACGGCGAU 1631 AUCGCCGUGGGGAUUGUCA 1632 1201CACGGCGAUGGGCGCUGCA 1633 UGCAGCGCCCAUCGCCGUG 1634 1210GGGCGCUGCAUCACCUGUA 1635 UACAGGUGAUGCAGCGCCC 1636 1211GGCGCUGCAUCACCUGUAU 1637 AUACAGGUGAUGCAGCGCC 1638 1213CGCUGCAUCACCUGUAUCU 1639 AGAUACAGGUGAUGCAGCG 1640 1216UGCAUCACCUGUAUCUAUU 1641 AAUAGAUACAGGUGAUGCA 1642 1217GCAUCACCUGUAUCUAUUA 1643 UAAUAGAUACAGGUGAUGC 1644 1220UCACCUGUAUCUAUUACCU 1645 AGGUAAUAGAUACAGGUGA 1646 1222ACCUGUAUCUAUUACCUGA 1647 UCAGGUAAUAGAUACAGGU 1648 1223CCUGUAUCUAUUACCUGAA 1649 UUCAGGUAAUAGAUACAGG 1650 1226GUAUCUAUUACCUGAAUCA 1651 UGAUUCAGGUAAUAGAUAC 1652 1228AUCUAUUACCUGAAUCAGA 1653 UCUGAUUCAGGUAAUAGAU 1654 1231UAUUACCUGAAUCAGAACU 1655 AGUUCUGAUUCAGGUAAUA 1656 1238UGAAUCAGAACUGGGACGU 1657 ACGUCCCAGUUCUGAUUCA 1658 1239GAAUCAGAACUGGGACGUU 1659 AACGUCCCAGUUCUGAUUC 1660 1240AAUCAGAACUGGGACGUUA 1661 UAACGUCCCAGUUCUGAUU 1662 1241AUCAGAACUGGGACGUUAA 1663 UUAACGUCCCAGUUCUGAU 1664 1244AGAACUGGGACGUUAAGGU 1665 ACCUUAACGUCCCAGUUCU 1666 1247ACUGGGACGUUAAGGUGCA 1667 UGCACCUUAACGUCCCAGU 1668 1248CUGGGACGUUAAGGUGCAU 1669 AUGCACCUUAACGUCCCAG 1670 1256UUAAGGUGCAUGGCGGCCU 1671 AGGCCGCCAUGCACCUUAA 1672 1259AGGUGCAUGGCGGCCUGCU 1673 AGCAGGCCGCCAUGCACCU 1674 1294GGCCGGCCCGUGGUAGCCA 1675 UGGCUACCACGGGCCGGCC 1676 1295GCCGGCCCGUGGUAGCCAA 1677 UUGGCUACCACGGGCCGGC 1678 1297CGGCCCGUGGUAGCCAACA 1679 UGUUGGCUACCACGGGCCG 1680 1298GGCCCGUGGUAGCCAACAU 1681 AUGUUGGCUACCACGGGCC 1682 1309GCCAACAUCGAGCCACUCU 1683 AGAGUGGCUCGAUGUUGGC 1684 1310CCAACAUCGAGCCACUCUU 1685 AAGAGUGGCUCGAUGUUGG 1686 1311CAACAUCGAGCCACUCUUU 1687 AAAGAGUGGCUCGAUGUUG 1688 1313ACAUCGAGCCACUCUUUGA 1689 UCAAAGAGUGGCUCGAUGU 1690 1318GAGCCACUCUUUGACCGGU 1691 ACCGGUCAAAGAGUGGCUC 1692 1319AGCCACUCUUUGACCGGUU 1693 AACCGGUCAAAGAGUGGCU 1694 1322CACUCUUUGACCGGUUGCU 1695 AGCAACCGGUCAAAGAGUG 1696 1324CUCUUUGACCGGUUGCUCA 1697 UGAGCAACCGGUCAAAGAG 1698 1325UCUUUGACCGGUUGCUCAU 1699 AUGAGCAACCGGUCAAAGA 1700 1326CUUUGACCGGUUGCUCAUU 1701 AAUGAGCAACCGGUCAAAG 1702 1327UUUGACCGGUUGCUCAUUU 1703 AAAUGAGCAACCGGUCAAA 1704 1330GACCGGUUGCUCAUUUUCU 1705 AGAAAAUGAGCAACCGGUC 1706 1346UCUGGUCUGACCGGCGGAA 1707 UUCCGCCGGUCAGACCAGA 1708 1352CUGACCGGCGGAACCCCCA 1709 UGGGGGUUCCGCCGGUCAG 1710 1355ACCGGCGGAACCCCCACGA 1711 UCGUGGGGGUUCCGCCGGU 1712 1358GGCGGAACCCCCACGAGGU 1713 ACCUCGUGGGGGUUCCGCC 1714 1361GGAACCCCCACGAGGUGAA 1715 UUCACCUCGUGGGGGUUCC 1716 1370ACGAGGUGAAGCCAGCCUA 1717 UAGGCUGGCUUCACCUCGU 1718 1375GUGAAGCCAGCCUAUGCCA 1719 UGGCAUAGGCUGGCUUCAC 1720 1381CCAGCCUAUGCCACCAGGU 1721 ACCUGGUGGCAUAGGCUGG 1722 1387UAUGCCACCAGGUACGCCA 1723 UGGCGUACCUGGUGGCAUA 1724 1388AUGCCACCAGGUACGCCAU 1725 AUGGCGUACCUGGUGGCAU 1726 1394CCAGGUACGCCAUCACUGU 1727 ACAGUGAUGGCGUACCUGG 1728 1396AGGUACGCCAUCACUGUCU 1729 AGACAGUGAUGGCGUACCU 1730 1401CGCCAUCACUGUCUGGUAU 1731 AUACCAGACAGUGAUGGCG 1732 1403CCAUCACUGUCUGGUAUUU 1733 AAAUACCAGACAGUGAUGG 1734 1438GCAGCAGCCAAAGACAAGU 1735 ACUUGUCUUUGGCUGCUGC 1736 1440AGCAGCCAAAGACAAGUAU 1737 AUACUUGUCUUUGGCUGCU 1738 1442CAGCCAAAGACAAGUAUCA 1739 UGAUACUUGUCUUUGGCUG 1740 1446CAAAGACAAGUAUCAGCUA 1741 UAGCUGAUACUUGUCUUUG 1742 1449AGACAAGUAUCAGCUAGCA 1743 UGCUAGCUGAUACUUGUCU 1744 1450GACAAGUAUCAGCUAGCAU 1745 AUGCUAGCUGAUACUUGUC 1746 1452CAAGUAUCAGCUAGCAUCA 1747 UGAUGCUAGCUGAUACUUG 1748 1455GUAUCAGCUAGCAUCAGGA 1749 UCCUGAUGCUAGCUGAUAC 1750 1457AUCAGCUAGCAUCAGGACA 1751 UGUCCUGAUGCUAGCUGAU 1752 1459CAGCUAGCAUCAGGACAGA 1753 UCUGUCCUGAUGCUAGCUG 1754 1461GCUAGCAUCAGGACAGAAA 1755 UUUCUGUCCUGAUGCUAGC 1756 1476GAAAGGUGUCCAAGUACCU 1757 AGGUACUUGGACACCUUUC 1758 1482UGUCCAAGUACCUGUAUCA 1759 UGAUACAGGUACUUGGACA 1760 1504CCGCCUACGCCCACCUAGU 1761 ACUAGGUGGGCGUAGGCGG 1762 1509UACGCCCACCUAGUGGCCA 1763 UGGCCACUAGGUGGGCGUA 1764 1517CCUAGUGGCCAGUCCCAGA 1765 UCUGGGACUGGCCACUAGG 1766 1538CGCAUGGCAGACAGCUUAA 1767 UUAAGCUGUCUGCCAUGCG 1768 1539GCAUGGCAGACAGCUUAAA 1769 UUUAAGCUGUCUGCCAUGC 1770 1542UGGCAGACAGCUUAAAUGA 1771 UCAUUUAAGCUGUCUGCCA 1772 1544GCAGACAGCUUAAAUGACU 1773 AGUCAUUUAAGCUGUCUGC 1774 1674AGGAGGAGAAGAGACCUUU 1775 AAAGGUCUCUUCUCCUCCU 1776 1684GAGACCUUUGCUGCCCCAU 1777 AUGGGGCAGCAAAGGUCUC 1778 1702UCAUGGGGGCUGGGGUUGU 1779 ACAACCCCAGCCCCCAUGA 1780 1741GUGGAGGCCACCGUUACCA 1781 UGGUAACGGUGGCCUCCAC 1782 1742UGGAGGCCACCGUUACCAA 1783 UUGGUAACGGUGGCCUCCA 1784 1744GAGGCCACCGUUACCAACU 1785 AGUUGGUAACGGUGGCCUC 1786 1746GGCCACCGUUACCAACUGA 1787 UCAGUUGGUAACGGUGGCC 1788 1774CCUGGGUCCUACCCUGUCU 1789 AGACAGGGUAGGACCCAGG 1790 1779GUCCUACCCUGUCUGGUCA 1791 UGACCAGACAGGGUAGGAC 1792 1782CUACCCUGUCUGGUCAUGA 1793 UCAUGACCAGACAGGGUAG 1794 1787CUGUCUGGUCAUGACCCCA 1795 UGGGGUCAUGACCAGACAG 1796 1788UGUCUGGUCAUGACCCCAU 1797 AUGGGGUCAUGACCAGACA 1798 1789GUCUGGUCAUGACCCCAUU 1799 AAUGGGGUCAUGACCAGAC 1800 1798UGACCCCAUUAGGUAUGGA 1801 UCCAUACCUAAUGGGGUCA 1802 1800ACCCCAUUAGGUAUGGAGA 1803 UCUCCAUACCUAAUGGGGU 1804 1807UAGGUAUGGAGAGCUGGGA 1805 UCCCAGCUCUCCAUACCUA 1806 1820CUGGGAGGAGGCAUUGUCA 1807 UGACAAUGCCUCCUCCCAG 1808 1823GGAGGAGGCAUUGUCACUU 1809 AAGUGACAAUGCCUCCUCC 1810 1827GAGGCAUUGUCACUUCCCA 1811 UGGGAAGUGACAAUGCCUC 1812 1830GCAUUGUCACUUCCCACCA 1813 UGGUGGGAAGUGACAAUGC 1814 1856GGACUUGGGGUUGAGGUGA 1815 UCACCUCAACCCCAAGUCC 1816 1858ACUUGGGGUUGAGGUGAGU 1817 ACUCACCUCAACCCCAAGU 1818 1861UGGGGUUGAGGUGAGUCAU 1819 AUGACUCACCUCAACCCCA 1820 1866UUGAGGUGAGUCAUGGCCU 1821 AGGCCAUGACUCACCUCAA 1822 1868GAGGUGAGUCAUGGCCUCU 1823 AGAGGCCAUGACUCACCUC 1824 1872UGAGUCAUGGCCUCUUGCU 1825 AGCAAGAGGCCAUGACUCA 1826 1876UCAUGGCCUCUUGCUGGCA 1827 UGCCAGCAAGAGGCCAUGA 1828 1878AUGGCCUCUUGCUGGCAAU 1829 AUUGCCAGCAAGAGGCCAU 1830 1883CUCUUGCUGGCAAUGGGGU 1831 ACCCCAUUGCCAGCAAGAG 1832 1893CAAUGGGGUGGGAGGAGUA 1833 UACUCCUCCCACCCCAUUG 1834 1902GGGAGGAGUACCCCCAAGU 1835 ACUUGGGGGUACUCCUCCC 1836 1905AGGAGUACCCCCAAGUCCU 1837 AGGACUUGGGGGUACUCCU 1838 1931CUCCAGCCUGGAAUGUGAA 1839 UUCACAUUCCAGGCUGGAG 1840 1933CCAGCCUGGAAUGUGAAGU 1841 ACUUCACAUUCCAGGCUGG 1842 1942AAUGUGAAGUGACUCCCCA 1843 UGGGGAGUCACUUCACAUU 1844 1964CCUUUGGCCAUGGCAGGCA 1845 UGCCUGCCAUGGCCAAAGG 1846 1973AUGGCAGGCACCUUUUGGA 1847 UCCAAAAGGUGCCUGCCAU 1848 1980GCACCUUUUGGACUGGGCU 1849 AGCCCAGUCCAAAAGGUGC 1850 2001CACUGCUUGGGCAGAGUAA 1851 UUACUCUGCCCAAGCAGUG 1852 2002ACUGCUUGGGCAGAGUAAA 1853 UUUACUCUGCCCAAGCAGU 1854 2003CUGCUUGGGCAGAGUAAAA 1855 UUUUACUCUGCCCAAGCAG 1856 2006CUUGGGCAGAGUAAAAGGU 1857 ACCUUUUACUCUGCCCAAG 1858 2010GGCAGAGUAAAAGGUGCCA 1859 UGGCACCUUUUACUCUGCC 1860 2077CCUCAGAGCUGCAAAAAAA 1861 UUUUUUUGCAGCUCUGAGG 1862

TABLE 6C  Human EGNL3 Single Strands and Duplex Sequences SEQ SEQ SenseID Antisense ID Start Sequence (5′ to 3′) NO. Sequence (5′ to 3′) NO. 6UGGCCGCAGUCGCGGCAGU 1863 ACUGCCGCGACUGCGGCCA 1864 35 CAUCCCCAAAAGGCGCCCU1865 AGGGCGCCUUUUGGGGAUG 1866 41 CAAAAGGCGCCCUCCGACU 1867AGUCGGAGGGCGCCUUUUG 1868 53 UCCGACUCCUUGCGCCGCA 1869 UGCGGCGCAAGGAGUCGGA1870 58 CUCCUUGCGCCGCACUGCU 1871 AGCAGUGCGGCGCAAGGAG 1872 75CUCGCCGGGCCAGUCCGGA 1873 UCCGGACUGGCCCGGCGAG 1874 76 UCGCCGGGCCAGUCCGGAA1875 UUCCGGACUGGCCCGGCGA 1876 77 CGCCGGGCCAGUCCGGAAA 1877UUUCCGGACUGGCCCGGCG 1878 85 CAGUCCGGAAACGGGUCGU 1879 ACGACCCGUUUCCGGACUG1880 88 UCCGGAAACGGGUCGUGGA 1881 UCCACGACCCGUUUCCGGA 1882 99GUCGUGGAGCUCCGCACCA 1883 UGGUGCGGAGCUCCACGAC 1884 101CGUGGAGCUCCGCACCACU 1885 AGUGGUGCGGAGCUCCACG 1886 107GCUCCGCACCACUCCCGCU 1887 AGCGGGAGUGGUGCGGAGC 1888 111CGCACCACUCCCGCUGGUU 1889 AACCAGCGGGAGUGGUGCG 1890 123GCUGGUUCCCGAAGGCAGA 1891 UCUGCCUUCGGGAACCAGC 1892 129UCCCGAAGGCAGAUCCCUU 1893 AAGGGAUCUGCCUUCGGGA 1894 138CAGAUCCCUUCUCCCGAGA 1895 UCUCGGGAGAAGGGAUCUG 1896 140GAUCCCUUCUCCCGAGAGU 1897 ACUCUCGGGAGAAGGGAUC 1898 141AUCCCUUCUCCCGAGAGUU 1899 AACUCUCGGGAGAAGGGAU 1900 145CUUCUCCCGAGAGUUGCGA 1901 UCGCAACUCUCGGGAGAAG 1902 147UCUCCCGAGAGUUGCGAGA 1903 UCUCGCAACUCUCGGGAGA 1904 148CUCCCGAGAGUUGCGAGAA 1905 UUCUCGCAACUCUCGGGAG 1906 149UCCCGAGAGUUGCGAGAAA 1907 UUUCUCGCAACUCUCGGGA 1908 151CCGAGAGUUGCGAGAAACU 1909 AGUUUCUCGCAACUCUCGG 1910 152CGAGAGUUGCGAGAAACUU 1911 AAGUUUCUCGCAACUCUCG 1912 153GAGAGUUGCGAGAAACUUU 1913 AAAGUUUCUCGCAACUCUC 1914 158UUGCGAGAAACUUUCCCUU 1915 AAGGGAAAGUUUCUCGCAA 1916 160GCGAGAAACUUUCCCUUGU 1917 ACAAGGGAAAGUUUCUCGC 1918 189GCAGCGGCUCGGGUACCGU 1919 ACGGUACCCGAGCCGCUGC 1920 206GUGGCAGCCGCAGGUUUCU 1921 AGAAACCUGCGGCUGCCAC 1922 208GGCAGCCGCAGGUUUCUGA 1923 UCAGAAACCUGCGGCUGCC 1924 209GCAGCCGCAGGUUUCUGAA 1925 UUCAGAAACCUGCGGCUGC 1926 245CGCGCCUCGGCUUCGCGCU 1927 AGCGCGAAGCCGAGGCGCG 1928 250CUCGGCUUCGCGCUCGUGU 1929 ACACGAGCGCGAAGCCGAG 1930 251UCGGCUUCGCGCUCGUGUA 1931 UACACGAGCGCGAAGCCGA 1932 253GGCUUCGCGCUCGUGUAGA 1933 UCUACACGAGCGCGAAGCC 1934 254GCUUCGCGCUCGUGUAGAU 1935 AUCUACACGAGCGCGAAGC 1936 257UCGCGCUCGUGUAGAUCGU 1937 ACGAUCUACACGAGCGCGA 1938 258CGCGCUCGUGUAGAUCGUU 1939 AACGAUCUACACGAGCGCG 1940 262CUCGUGUAGAUCGUUCCCU 1941 AGGGAACGAUCUACACGAG 1942 270GAUCGUUCCCUCUCUGGUU 1943 AACCAGAGAGGGAACGAUC 1944 273CGUUCCCUCUCUGGUUGCA 1945 UGCAACCAGAGAGGGAACG 1946 277CCCUCUCUGGUUGCACGCU 1947 AGCGUGCAACCAGAGAGGG 1948 282UCUGGUUGCACGCUGGGGA 1949 UCCCCAGCGUGCAACCAGA 1950 283CUGGUUGCACGCUGGGGAU 1951 AUCCCCAGCGUGCAACCAG 1952 295UGGGGAUCCCGGACCUCGA 1953 UCGAGGUCCGGGAUCCCCA 1954 296GGGGAUCCCGGACCUCGAU 1955 AUCGAGGUCCGGGAUCCCC 1956 299GAUCCCGGACCUCGAUUCU 1957 AGAAUCGAGGUCCGGGAUC 1958 307ACCUCGAUUCUGCGGGCGA 1959 UCGCCCGCAGAAUCGAGGU 1960 309CUCGAUUCUGCGGGCGAGA 1961 UCUCGCCCGCAGAAUCGAG 1962 355ACCUGGAGAAAAUUGCCCU 1963 AGGGCAAUUUUCUCCAGGU 1964 367UUGCCCUGGAGUACAUCGU 1965 ACGAUGUACUCCAGGGCAA 1966 376AGUACAUCGUGCCCUGUCU 1967 AGACAGGGCACGAUGUACU 1968 382UCGUGCCCUGUCUGCACGA 1969 UCGUGCAGACAGGGCACGA 1970 390UGUCUGCACGAGGUGGGCU 1971 AGCCCACCUCGUGCAGACA 1972 451GCGUCCUGGAGCGCGUCAA 1973 UUGACGCGCUCCAGGACGC 1974 521CGCCGGCGUCUCCAAGCGA 1975 UCGCUUGGAGACGCCGGCG 1976 526GCGUCUCCAAGCGACACCU 1977 AGGUGUCGCUUGGAGACGC 1978 538GACACCUGCGGGGCGACCA 1979 UGGUCGCCCCGCAGGUGUC 1980 540CACCUGCGGGGCGACCAGA 1981 UCUGGUCGCCCCGCAGGUG 1982 559UCACGUGGAUCGGGGGCAA 1983 UUGCCCCCGAUCCACGUGA 1984 565GGAUCGGGGGCAACGAGGA 1985 UCCUCGUUGCCCCCGAUCC 1986 619UCGACAGGCUGGUCCUCUA 1987 UAGAGGACCAGCCUGUCGA 1988 621GACAGGCUGGUCCUCUACU 1989 AGUAGAGGACCAGCCUGUC 1990 627CUGGUCCUCUACUGCGGGA 1991 UCCCGCAGUAGAGGACCAG 1992 643GGAGCCGGCUGGGCAAAUA 1993 UAUUUGCCCAGCCGGCUCC 1994 646GCCGGCUGGGCAAAUACUA 1995 UAGUAUUUGCCCAGCCGGC 1996 649GGCUGGGCAAAUACUACGU 1997 ACGUAGUAUUUGCCCAGCC 1998 651CUGGGCAAAUACUACGUCA 1999 UGACGUAGUAUUUGCCCAG 2000 652UGGGCAAAUACUACGUCAA 2001 UUGACGUAGUAUUUGCCCA 2002 655GCAAAUACUACGUCAAGGA 2003 UCCUUGACGUAGUAUUUGC 2004 662CUACGUCAAGGAGAGGUCU 2005 AGACCUCUCCUUGACGUAG 2006 663UACGUCAAGGAGAGGUCUA 2007 UAGACCUCUCCUUGACGUA 2008 668CAAGGAGAGGUCUAAGGCA 2009 UGCCUUAGACCUCUCCUUG 2010 673AGAGGUCUAAGGCAAUGGU 2011 ACCAUUGCCUUAGACCUCU 2012 678UCUAAGGCAAUGGUGGCUU 2013 AAGCCACCAUUGCCUUAGA 2014 681AAGGCAAUGGUGGCUUGCU 2015 AGCAAGCCACCAUUGCCUU 2016 682AGGCAAUGGUGGCUUGCUA 2017 UAGCAAGCCACCAUUGCCU 2018 683GGCAAUGGUGGCUUGCUAU 2019 AUAGCAAGCCACCAUUGCC 2020 690GUGGCUUGCUAUCCGGGAA 2021 UUCCCGGAUAGCAAGCCAC 2022 691UGGCUUGCUAUCCGGGAAA 2023 UUUCCCGGAUAGCAAGCCA 2024 692GGCUUGCUAUCCGGGAAAU 2025 AUUUCCCGGAUAGCAAGCC 2026 696UGCUAUCCGGGAAAUGGAA 2027 UUCCAUUUCCCGGAUAGCA 2028 702CCGGGAAAUGGAACAGGUU 2029 AACCUGUUCCAUUUCCCGG 2030 704GGGAAAUGGAACAGGUUAU 2031 AUAACCUGUUCCAUUUCCC 2032 712GAACAGGUUAUGUUCGCCA 2033 UGGCGAACAUAACCUGUUC 2034 715CAGGUUAUGUUCGCCACGU 2035 ACGUGGCGAACAUAACCUG 2036 718GUUAUGUUCGCCACGUGGA 2037 UCCACGUGGCGAACAUAAC 2038 720UAUGUUCGCCACGUGGACA 2039 UGUCCACGUGGCGAACAUA 2040 721AUGUUCGCCACGUGGACAA 2041 UUGUCCACGUGGCGAACAU 2042 726CGCCACGUGGACAACCCCA 2043 UGGGGUUGUCCACGUGGCG 2044 731CGUGGACAACCCCAACGGU 2045 ACCGUUGGGGUUGUCCACG 2046 734GGACAACCCCAACGGUGAU 2047 AUCACCGUUGGGGUUGUCC 2048 737CAACCCCAACGGUGAUGGU 2049 ACCAUCACCGUUGGGGUUG 2050 741CCCAACGGUGAUGGUCGCU 2051 AGCGACCAUCACCGUUGGG 2052 744AACGGUGAUGGUCGCUGCA 2053 UGCAGCGACCAUCACCGUU 2054 765ACCUGCAUCUACUAUCUGA 2055 UCAGAUAGUAGAUGCAGGU 2056 766CCUGCAUCUACUAUCUGAA 2057 UUCAGAUAGUAGAUGCAGG 2058 787AGAAUUGGGAUGCCAAGCU 2059 AGCUUGGCAUCCCAAUUCU 2060 788GAAUUGGGAUGCCAAGCUA 2061 UAGCUUGGCAUCCCAAUUC 2062 790AUUGGGAUGCCAAGCUACA 2063 UGUAGCUUGGCAUCCCAAU 2064 802AGCUACAUGGUGGGAUCCU 2065 AGGAUCCCACCAUGUAGCU 2066 808AUGGUGGGAUCCUGCGGAU 2067 AUCCGCAGGAUCCCACCAU 2068 809UGGUGGGAUCCUGCGGAUA 2069 UAUCCGCAGGAUCCCACCA 2070 810GGUGGGAUCCUGCGGAUAU 2071 AUAUCCGCAGGAUCCCACC 2072 811GUGGGAUCCUGCGGAUAUU 2073 AAUAUCCGCAGGAUCCCAC 2074 812UGGGAUCCUGCGGAUAUUU 2075 AAAUAUCCGCAGGAUCCCA 2076 815GAUCCUGCGGAUAUUUCCA 2077 UGGAAAUAUCCGCAGGAUC 2078 817UCCUGCGGAUAUUUCCAGA 2079 UCUGGAAAUAUCCGCAGGA 2080 822CGGAUAUUUCCAGAGGGGA 2081 UCCCCUCUGGAAAUAUCCG 2082 833AGAGGGGAAAUCAUUCAUA 2083 UAUGAAUGAUUUCCCCUCU 2084 836GGGGAAAUCAUUCAUAGCA 2085 UGCUAUGAAUGAUUUCCCC 2086 839GAAAUCAUUCAUAGCAGAU 2087 AUCUGCUAUGAAUGAUUUC 2088 858GUGGAGCCCAUUUUUGACA 2089 UGUCAAAAAUGGGCUCCAC 2090 860GGAGCCCAUUUUUGACAGA 2091 UCUGUCAAAAAUGGGCUCC 2092 862AGCCCAUUUUUGACAGACU 2093 AGUCUGUCAAAAAUGGGCU 2094 868UUUUUGACAGACUCCUGUU 2095 AACAGGAGUCUGUCAAAAA 2096 871UUGACAGACUCCUGUUCUU 2097 AAGAACAGGAGUCUGUCAA 2098 873GACAGACUCCUGUUCUUCU 2099 AGAAGAACAGGAGUCUGUC 2100 881CCUGUUCUUCUGGUCAGAU 2101 AUCUGACCAGAAGAACAGG 2102 884GUUCUUCUGGUCAGAUCGU 2103 ACGAUCUGACCAGAAGAAC 2104 885UUCUUCUGGUCAGAUCGUA 2105 UACGAUCUGACCAGAAGAA 2106 888UUCUGGUCAGAUCGUAGGA 2107 UCCUACGAUCUGACCAGAA 2108 889UCUGGUCAGAUCGUAGGAA 2109 UUCCUACGAUCUGACCAGA 2110 893GUCAGAUCGUAGGAACCCA 2111 UGGGUUCCUACGAUCUGAC 2112 895CAGAUCGUAGGAACCCACA 2113 UGUGGGUUCCUACGAUCUG 2114 898AUCGUAGGAACCCACACGA 2115 UCGUGUGGGUUCCUACGAU 2116 899UCGUAGGAACCCACACGAA 2117 UUCGUGUGGGUUCCUACGA 2118 901GUAGGAACCCACACGAAGU 2119 ACUUCGUGUGGGUUCCUAC 2120 904GGAACCCACACGAAGUGCA 2121 UGCACUUCGUGUGGGUUCC 2122 917AGUGCAGCCCUCUUACGCA 2123 UGCGUAAGAGGGCUGCACU 2124 918GUGCAGCCCUCUUACGCAA 2125 UUGCGUAAGAGGGCUGCAC 2126 921CAGCCCUCUUACGCAACCA 2127 UGGUUGCGUAAGAGGGCUG 2128 923GCCCUCUUACGCAACCAGA 2129 UCUGGUUGCGUAAGAGGGC 2130 926CUCUUACGCAACCAGAUAU 2131 AUAUCUGGUUGCGUAAGAG 2132 929UUACGCAACCAGAUAUGCU 2133 AGCAUAUCUGGUUGCGUAA 2134 933GCAACCAGAUAUGCUAUGA 2135 UCAUAGCAUAUCUGGUUGC 2136 935AACCAGAUAUGCUAUGACU 2137 AGUCAUAGCAUAUCUGGUU 2138 937CCAGAUAUGCUAUGACUGU 2139 ACAGUCAUAGCAUAUCUGG 2140 942UAUGCUAUGACUGUCUGGU 2141 ACCAGACAGUCAUAGCAUA 2142 943AUGCUAUGACUGUCUGGUA 2143 UACCAGACAGUCAUAGCAU 2144 946CUAUGACUGUCUGGUACUU 2145 AAGUACCAGACAGUCAUAG 2146 955UCUGGUACUUUGAUGCUGA 2147 UCAGCAUCAAAGUACCAGA 2148 974AGAAAGGGCAGAAGCCAAA 2149 UUUGGCUUCUGCCCUUUCU 2150 978AGGGCAGAAGCCAAAAAGA 2151 UCUUUUUGGCUUCUGCCCU 2152 995GAAAUUCAGGAAUUUAACU 2153 AGUUAAAUUCCUGAAUUUC 2154 996AAAUUCAGGAAUUUAACUA 2155 UAGUUAAAUUCCUGAAUUU 2156 999UUCAGGAAUUUAACUAGGA 2157 UCCUAGUUAAAUUCCUGAA 2158 1000UCAGGAAUUUAACUAGGAA 2159 UUCCUAGUUAAAUUCCUGA 2160 1002AGGAAUUUAACUAGGAAAA 2161 UUUUCCUAGUUAAAUUCCU 2162 1007UUUAACUAGGAAAACUGAA 2163 UUCAGUUUUCCUAGUUAAA 2164 1015GGAAAACUGAAUCUGCCCU 2165 AGGGCAGAUUCAGUUUUCC 2166 1019AACUGAAUCUGCCCUCACU 2167 AGUGAGGGCAGAUUCAGUU 2168 1022UGAAUCUGCCCUCACUGAA 2169 UUCAGUGAGGGCAGAUUCA 2170 1032CUCACUGAAGACUGACCGU 2171 ACGGUCAGUCUUCAGUGAG 2172 1037UGAAGACUGACCGUGCUCU 2173 AGAGCACGGUCAGUCUUCA 2174 1039AAGACUGACCGUGCUCUGA 2175 UCAGAGCACGGUCAGUCUU 2176 1040AGACUGACCGUGCUCUGAA 2177 UUCAGAGCACGGUCAGUCU 2178 1044UGACCGUGCUCUGAAAUCU 2179 AGAUUUCAGAGCACGGUCA 2180 1052CUCUGAAAUCUGCUGGCCU 2181 AGGCCAGCAGAUUUCAGAG 2182 1053UCUGAAAUCUGCUGGCCUU 2183 AAGGCCAGCAGAUUUCAGA 2184 1060UCUGCUGGCCUUGUUCAUU 2185 AAUGAACAAGGCCAGCAGA 2186 1062UGCUGGCCUUGUUCAUUUU 2187 AAAAUGAACAAGGCCAGCA 2188 1071UGUUCAUUUUAGUAACGGU 2189 ACCGUUACUAAAAUGAACA 2190 1072GUUCAUUUUAGUAACGGUU 2191 AACCGUUACUAAAAUGAAC 2192 1075CAUUUUAGUAACGGUUCCU 2193 AGGAACCGUUACUAAAAUG 2194 1078UUUAGUAACGGUUCCUGAA 2195 UUCAGGAACCGUUACUAAA 2196 1079UUAGUAACGGUUCCUGAAU 2197 AUUCAGGAACCGUUACUAA 2198 1080UAGUAACGGUUCCUGAAUU 2199 AAUUCAGGAACCGUUACUA 2200 1082GUAACGGUUCCUGAAUUCU 2201 AGAAUUCAGGAACCGUUAC 2202 1084AACGGUUCCUGAAUUCUCU 2203 AGAGAAUUCAGGAACCGUU 2204 1088GUUCCUGAAUUCUCUUAAA 2205 UUUAAGAGAAUUCAGGAAC 2206 1092CUGAAUUCUCUUAAAUUCU 2207 AGAAUUUAAGAGAAUUCAG 2208 1112UGAGAUCCAAAGAUGGCCU 2209 AGGCCAUCUUUGGAUCUCA 2210 1115GAUCCAAAGAUGGCCUCUU 2211 AAGAGGCCAUCUUUGGAUC 2212 1119CAAAGAUGGCCUCUUCAGU 2213 ACUGAAGAGGCCAUCUUUG 2214 1137UGACAACAAUCUCCCUGCU 2215 AGCAGGGAGAUUGUUGUCA 2216 1141AACAAUCUCCCUGCUACUU 2217 AAGUAGCAGGGAGAUUGUU 2218 1148UCCCUGCUACUUCUUGCAU 2219 AUGCAAGAAGUAGCAGGGA 2220 1151CUGCUACUUCUUGCAUCCU 2221 AGGAUGCAAGAAGUAGCAG 2222 1152UGCUACUUCUUGCAUCCUU 2223 AAGGAUGCAAGAAGUAGCA 2224 1176CCCUGUCUUGUGUGUGGUA 2225 UACCACACACAAGACAGGG 2226 1181UCUUGUGUGUGGUACUUCA 2227 UGAAGUACCACACACAAGA 2228 1182CUUGUGUGUGGUACUUCAU 2229 AUGAAGUACCACACACAAG 2230 1186UGUGUGGUACUUCAUGUUU 2231 AAACAUGAAGUACCACACA 2232 1194ACUUCAUGUUUUCUUGCCA 2233 UGGCAAGAAAACAUGAAGU 2234 1201GUUUUCUUGCCAAGACUGU 2235 ACAGUCUUGGCAAGAAAAC 2236 1204UUCUUGCCAAGACUGUGUU 2237 AACACAGUCUUGGCAAGAA 2238 1218GUGUUGAUCUUCAGAUACU 2239 AGUAUCUGAAGAUCAACAC 2240 1222UGAUCUUCAGAUACUCUCU 2241 AGAGAGUAUCUGAAGAUCA 2242 1228UCAGAUACUCUCUUUGCCA 2243 UGGCAAAGAGAGUAUCUGA 2244 1230AGAUACUCUCUUUGCCAGA 2245 UCUGGCAAAGAGAGUAUCU 2246 1233UACUCUCUUUGCCAGAUGA 2247 UCAUCUGGCAAAGAGAGUA 2248 1234ACUCUCUUUGCCAGAUGAA 2249 UUCAUCUGGCAAAGAGAGU 2250 1241UUGCCAGAUGAAGUUACUU 2251 AAGUAACUUCAUCUGGCAA 2252 1245CAGAUGAAGUUACUUGCUA 2253 UAGCAAGUAACUUCAUCUG 2254 1246AGAUGAAGUUACUUGCUAA 2255 UUAGCAAGUAACUUCAUCU 2256 1248AUGAAGUUACUUGCUAACU 2257 AGUUAGCAAGUAACUUCAU 2258 1251AAGUUACUUGCUAACUCCA 2259 UGGAGUUAGCAAGUAACUU 2260 1255UACUUGCUAACUCCAGAAA 2261 UUUCUGGAGUUAGCAAGUA 2262 1260GCUAACUCCAGAAAUUCCU 2263 AGGAAUUUCUGGAGUUAGC 2264 1272AAUUCCUGCAGACAUCCUA 2265 UAGGAUGUCUGCAGGAAUU 2266 1274UUCCUGCAGACAUCCUACU 2267 AGUAGGAUGUCUGCAGGAA 2268 1287CCUACUCGGCCAGCGGUUU 2269 AAACCGCUGGCCGAGUAGG 2270 1288CUACUCGGCCAGCGGUUUA 2271 UAAACCGCUGGCCGAGUAG 2272 1291CUCGGCCAGCGGUUUACCU 2273 AGGUAAACCGCUGGCCGAG 2274 1294GGCCAGCGGUUUACCUGAU 2275 AUCAGGUAAACCGCUGGCC 2276 1295GCCAGCGGUUUACCUGAUA 2277 UAUCAGGUAAACCGCUGGC 2278 1297CAGCGGUUUACCUGAUAGA 2279 UCUAUCAGGUAAACCGCUG 2280 1298AGCGGUUUACCUGAUAGAU 2281 AUCUAUCAGGUAAACCGCU 2282 1299GCGGUUUACCUGAUAGAUU 2283 AAUCUAUCAGGUAAACCGC 2284 1303UUUACCUGAUAGAUUCGGU 2285 ACCGAAUCUAUCAGGUAAA 2286 1304UUACCUGAUAGAUUCGGUA 2287 UACCGAAUCUAUCAGGUAA 2288 1305UACCUGAUAGAUUCGGUAA 2289 UUACCGAAUCUAUCAGGUA 2290 1306ACCUGAUAGAUUCGGUAAU 2291 AUUACCGAAUCUAUCAGGU 2292 1307CCUGAUAGAUUCGGUAAUA 2293 UAUUACCGAAUCUAUCAGG 2294 1309UGAUAGAUUCGGUAAUACU 2295 AGUAUUACCGAAUCUAUCA 2296 1310GAUAGAUUCGGUAAUACUA 2297 UAGUAUUACCGAAUCUAUC 2298 1313AGAUUCGGUAAUACUAUCA 2299 UGAUAGUAUUACCGAAUCU 2300 1325ACUAUCAAGAGAAGAGCCU 2301 AGGCUCUUCUCUUGAUAGU 2302 1329UCAAGAGAAGAGCCUAGGA 2303 UCCUAGGCUCUUCUCUUGA 2304 1344AGGAGCACAGCGAGGGAAU 2305 AUUCCCUCGCUGUGCUCCU 2306 1346GAGCACAGCGAGGGAAUGA 2307 UCAUUCCCUCGCUGUGCUC 2308 1347AGCACAGCGAGGGAAUGAA 2309 UUCAUUCCCUCGCUGUGCU 2310 1350ACAGCGAGGGAAUGAACCU 2311 AGGUUCAUUCCCUCGCUGU 2312 1351CAGCGAGGGAAUGAACCUU 2313 AAGGUUCAUUCCCUCGCUG 2314 1352AGCGAGGGAAUGAACCUUA 2315 UAAGGUUCAUUCCCUCGCU 2316 1360AAUGAACCUUACUUGCACU 2317 AGUGCAAGUAAGGUUCAUU 2318 1361AUGAACCUUACUUGCACUU 2319 AAGUGCAAGUAAGGUUCAU 2320 1362UGAACCUUACUUGCACUUU 2321 AAAGUGCAAGUAAGGUUCA 2322 1367CUUACUUGCACUUUAUGUA 2323 UACAUAAAGUGCAAGUAAG 2324 1368UUACUUGCACUUUAUGUAU 2325 AUACAUAAAGUGCAAGUAA 2326 1375CACUUUAUGUAUACUUCCU 2327 AGGAAGUAUACAUAAAGUG 2328 1378UUUAUGUAUACUUCCUGAU 2329 AUCAGGAAGUAUACAUAAA 2330 1379UUAUGUAUACUUCCUGAUU 2331 AAUCAGGAAGUAUACAUAA 2332 1383GUAUACUUCCUGAUUUGAA 2333 UUCAAAUCAGGAAGUAUAC 2334 1384UAUACUUCCUGAUUUGAAA 2335 UUUCAAAUCAGGAAGUAUA 2336 1395AUUUGAAAGGAGGAGGUUU 2337 AAACCUCCUCCUUUCAAAU 2338 1397UUGAAAGGAGGAGGUUUGA 2339 UCAAACCUCCUCCUUUCAA 2340 1419GAAAAAAAUGGAGGUGGUA 2341 UACCACCUCCAUUUUUUUC 2342 1422AAAAAUGGAGGUGGUAGAU 2343 AUCUACCACCUCCAUUUUU 2344 1428GGAGGUGGUAGAUGCCACA 2345 UGUGGCAUCUACCACCUCC 2346 1436UAGAUGCCACAGAGAGGCA 2347 UGCCUCUCUGUGGCAUCUA 2348 1443CACAGAGAGGCAUCACGGA 2349 UCCGUGAUGCCUCUCUGUG 2350 1451GGCAUCACGGAAGCCUUAA 2351 UUAAGGCUUCCGUGAUGCC 2352 1453CAUCACGGAAGCCUUAACA 2353 UGUUAAGGCUUCCGUGAUG 2354 1456CACGGAAGCCUUAACAGCA 2355 UGCUGUUAAGGCUUCCGUG 2356 1476GAAACAGAGAAAUUUGUGU 2357 ACACAAAUUUCUCUGUUUC 2358 1487AUUUGUGUCAUCUGAACAA 2359 UUGUUCAGAUGACACAAAU 2360 1499UGAACAAUUUCCAGAUGUU 2361 AACAUCUGGAAAUUGUUCA 2362 1501AACAAUUUCCAGAUGUUCU 2363 AGAACAUCUGGAAAUUGUU 2364 1502ACAAUUUCCAGAUGUUCUU 2365 AAGAACAUCUGGAAAUUGU 2366 1504AAUUUCCAGAUGUUCUUAA 2367 UUAAGAACAUCUGGAAAUU 2368 1513AUGUUCUUAAUCCAGGGCU 2369 AGCCCUGGAUUAAGAACAU 2370 1534UGGGGUUUCUGGAGAAUUA 2371 UAAUUCUCCAGAAACCCCA 2372 1539UUUCUGGAGAAUUAUCACA 2373 UGUGAUAAUUCUCCAGAAA 2374 1543UGGAGAAUUAUCACAACCU 2375 AGGUUGUGAUAAUUCUCCA 2376 1544GGAGAAUUAUCACAACCUA 2377 UAGGUUGUGAUAAUUCUCC 2378 1545GAGAAUUAUCACAACCUAA 2379 UUAGGUUGUGAUAAUUCUC 2380 1546AGAAUUAUCACAACCUAAU 2381 AUUAGGUUGUGAUAAUUCU 2382 1548AAUUAUCACAACCUAAUGA 2383 UCAUUAGGUUGUGAUAAUU 2384 1552AUCACAACCUAAUGACAUU 2385 AAUGUCAUUAGGUUGUGAU 2386 1553UCACAACCUAAUGACAUUA 2387 UAAUGUCAUUAGGUUGUGA 2388 1559CCUAAUGACAUUAAUACCU 2389 AGGUAUUAAUGUCAUUAGG 2390 1561UAAUGACAUUAAUACCUCU 2391 AGAGGUAUUAAUGUCAUUA 2392 1565GACAUUAAUACCUCUAGAA 2393 UUCUAGAGGUAUUAAUGUC 2394 1571AAUACCUCUAGAAAGGGCU 2395 AGCCCUUUCUAGAGGUAUU 2396 1582AAAGGGCUGCUGUCAUAGU 2397 ACUAUGACAGCAGCCCUUU 2398 1584AGGGCUGCUGUCAUAGUGA 2399 UCACUAUGACAGCAGCCCU 2400 1585GGGCUGCUGUCAUAGUGAA 2401 UUCACUAUGACAGCAGCCC 2402 1587GCUGCUGUCAUAGUGAACA 2403 UGUUCACUAUGACAGCAGC 2404 1589UGCUGUCAUAGUGAACAAU 2405 AUUGUUCACUAUGACAGCA 2406 1594UCAUAGUGAACAAUUUAUA 2407 UAUAAAUUGUUCACUAUGA 2408 1595CAUAGUGAACAAUUUAUAA 2409 UUAUAAAUUGUUCACUAUG 2410 1610AUAAGUGUCCCAUGGGGCA 2411 UGCCCCAUGGGACACUUAU 2412 1620CAUGGGGCAGACACUCCUU 2413 AAGGAGUGUCUGCCCCAUG 2414 1621AUGGGGCAGACACUCCUUU 2415 AAAGGAGUGUCUGCCCCAU 2416 1623GGGGCAGACACUCCUUUUU 2417 AAAAAGGAGUGUCUGCCCC 2418 1624GGGCAGACACUCCUUUUUU 2419 AAAAAAGGAGUGUCUGCCC 2420 1636CUUUUUUCCCAGUCCUGCA 2421 UGCAGGACUGGGAAAAAAG 2422 1640UUUCCCAGUCCUGCAACCU 2423 AGGUUGCAGGACUGGGAAA 2424 1645CAGUCCUGCAACCUGGAUU 2425 AAUCCAGGUUGCAGGACUG 2426 1647GUCCUGCAACCUGGAUUUU 2427 AAAAUCCAGGUUGCAGGAC 2428 1653CAACCUGGAUUUUCUGCCU 2429 AGGCAGAAAAUCCAGGUUG 2430 1670CUCAGCCCCAUUUUGCUGA 2431 UCAGCAAAAUGGGGCUGAG 2432 1694AUGACUUUCUGAAUAAAGA 2433 UCUUUAUUCAGAAAGUCAU 2434 1695UGACUUUCUGAAUAAAGAU 2435 AUCUUUAUUCAGAAAGUCA 2436 1704GAAUAAAGAUGGCAACACA 2437 UGUGUUGCCAUCUUUAUUC 2438 1732CCAUUUUCAGUUCUUACCU 2439 AGGUAAGAACUGAAAAUGG 2440 1736UUUCAGUUCUUACCUGGGA 2441 UCCCAGGUAAGAACUGAAA 2442 1737UUCAGUUCUUACCUGGGAA 2443 UUCCCAGGUAAGAACUGAA 2444 1741GUUCUUACCUGGGAACCUA 2445 UAGGUUCCCAGGUAAGAAC 2446 1742UUCUUACCUGGGAACCUAA 2447 UUAGGUUCCCAGGUAAGAA 2448 1743UCUUACCUGGGAACCUAAU 2449 AUUAGGUUCCCAGGUAAGA 2450 1744CUUACCUGGGAACCUAAUU 2451 AAUUAGGUUCCCAGGUAAG 2452 1749CUGGGAACCUAAUUCCCCA 2453 UGGGGAAUUAGGUUCCCAG 2454 1751GGGAACCUAAUUCCCCAGA 2455 UCUGGGGAAUUAGGUUCCC 2456 1752GGAACCUAAUUCCCCAGAA 2457 UUCUGGGGAAUUAGGUUCC 2458 1757CUAAUUCCCCAGAAGCUAA 2459 UUAGCUUCUGGGGAAUUAG 2460 1758UAAUUCCCCAGAAGCUAAA 2461 UUUAGCUUCUGGGGAAUUA 2462 1759AAUUCCCCAGAAGCUAAAA 2463 UUUUAGCUUCUGGGGAAUU 2464 1760AUUCCCCAGAAGCUAAAAA 2465 UUUUUAGCUUCUGGGGAAU 2466 1763CCCCAGAAGCUAAAAAACU 2467 AGUUUUUUAGCUUCUGGGG 2468 1764CCCAGAAGCUAAAAAACUA 2469 UAGUUUUUUAGCUUCUGGG 2470 1776AAAACUAGACAUUAGUUGU 2471 ACAACUAAUGUCUAGUUUU 2472 1777AAACUAGACAUUAGUUGUU 2473 AACAACUAAUGUCUAGUUU 2474 1778AACUAGACAUUAGUUGUUU 2475 AAACAACUAAUGUCUAGUU 2476 1779ACUAGACAUUAGUUGUUUU 2477 AAAACAACUAAUGUCUAGU 2478 1782AGACAUUAGUUGUUUUGGU 2479 ACCAAAACAACUAAUGUCU 2480 1783GACAUUAGUUGUUUUGGUU 2481 AACCAAAACAACUAAUGUC 2482 1788UAGUUGUUUUGGUUGCUUU 2483 AAAGCAACCAAAACAACUA 2484 1791UUGUUUUGGUUGCUUUGUU 2485 AACAAAGCAACCAAAACAA 2486 1844AUAUCCCUGGUAGUUUUGU 2487 ACAAAACUACCAGGGAUAU 2488 1847UCCCUGGUAGUUUUGUGUU 2489 AACACAAAACUACCAGGGA 2490 1849CCUGGUAGUUUUGUGUUAA 2491 UUAACACAAAACUACCAGG 2492 1854UAGUUUUGUGUUAACCACU 2493 AGUGGUUAACACAAAACUA 2494 1861GUGUUAACCACUGAUAACU 2495 AGUUAUCAGUGGUUAACAC 2496 1863GUUAACCACUGAUAACUGU 2497 ACAGUUAUCAGUGGUUAAC 2498 1868CCACUGAUAACUGUGGAAA 2499 UUUCCACAGUUAUCAGUGG 2500 1870ACUGAUAACUGUGGAAAGA 2501 UCUUUCCACAGUUAUCAGU 2502 1882GGAAAGAGCUAGGUCUACU 2503 AGUAGACCUAGCUCUUUCC 2504 1888AGCUAGGUCUACUGAUAUA 2505 UAUAUCAGUAGACCUAGCU 2506 1890CUAGGUCUACUGAUAUACA 2507 UGUAUAUCAGUAGACCUAG 2508 1893GGUCUACUGAUAUACAAUA 2509 UAUUGUAUAUCAGUAGACC 2510 1894GUCUACUGAUAUACAAUAA 2511 UUAUUGUAUAUCAGUAGAC 2512 1895UCUACUGAUAUACAAUAAA 2513 UUUAUUGUAUAUCAGUAGA 2514 1897UACUGAUAUACAAUAAACA 2515 UGUUUAUUGUAUAUCAGUA 2516 1905UACAAUAAACAUGUGUGCA 2517 UGCACACAUGUUUAUUGUA 2518 1911AAACAUGUGUGCAUCUUGA 2519 UCAAGAUGCACACAUGUUU 2520 1915AUGUGUGCAUCUUGAACAA 2521 UUGUUCAAGAUGCACACAU 2522 1916UGUGUGCAUCUUGAACAAU 2523 AUUGUUCAAGAUGCACACA 2524 1917GUGUGCAUCUUGAACAAUU 2525 AAUUGUUCAAGAUGCACAC 2526 1922CAUCUUGAACAAUUUGAGA 2527 UCUCAAAUUGUUCAAGAUG 2528 1927UGAACAAUUUGAGAGGGGA 2529 UCCCCUCUCAAAUUGUUCA 2530 1930ACAAUUUGAGAGGGGAGGU 2531 ACCUCCCCUCUCAAAUUGU 2532 1954UGGAAAUGUGGGUGUUCCU 2533 AGGAACACCCACAUUUCCA 2534 1958AAUGUGGGUGUUCCUGUUU 2535 AAACAGGAACACCCACAUU 2536 1962UGGGUGUUCCUGUUUUUUU 2537 AAAAAAACAGGAACACCCA 2538 2007UUAAUGAGCUCACCCUUUA 2539 UAAAGGGUGAGCUCAUUAA 2540 2008UAAUGAGCUCACCCUUUAA 2541 UUAAAGGGUGAGCUCAUUA 2542 2010AUGAGCUCACCCUUUAACA 2543 UGUUAAAGGGUGAGCUCAU 2544 2012GAGCUCACCCUUUAACACA 2545 UGUGUUAAAGGGUGAGCUC 2546 2014GCUCACCCUUUAACACAAA 2547 UUUGUGUUAAAGGGUGAGC 2548 2016UCACCCUUUAACACAAAAA 2549 UUUUUGUGUUAAAGGGUGA 2550 2017CACCCUUUAACACAAAAAA 2551 UUUUUUGUGUUAAAGGGUG 2552 2021CUUUAACACAAAAAAAGCA 2553 UGCUUUUUUUGUGUUAAAG 2554 2028ACAAAAAAAGCAAGGUGAU 2555 AUCACCUUGCUUUUUUUGU 2556 2044GAUGUAUUUUAAAAAAGGA 2557 UCCUUUUUUAAAAUACAUC 2558 2060GGAAGUGGAAAUAAAAAAA 2559 UUUUUUUAUUUCCACUUCC 2560 2072AAAAAAAUCUCAAAGCUAU 2561 AUAGCUUUGAGAUUUUUUU 2562 2073AAAAAAUCUCAAAGCUAUU 2563 AAUAGCUUUGAGAUUUUUU 2564 2081UCAAAGCUAUUUGAGUUCU 2565 AGAACUCAAAUAGCUUUGA 2566 2084AAGCUAUUUGAGUUCUCGU 2567 ACGAGAACUCAAAUAGCUU 2568 2086GCUAUUUGAGUUCUCGUCU 2569 AGACGAGAACUCAAAUAGC 2570 2098CUCGUCUGUCCCUAGCAGU 2571 ACUGCUAGGGACAGACGAG 2572 2100CGUCUGUCCCUAGCAGUCU 2573 AGACUGCUAGGGACAGACG 2574 2105GUCCCUAGCAGUCUUUCUU 2575 AAGAAAGACUGCUAGGGAC 2576 2121CUUCAGCUCACUUGGCUCU 2577 AGAGCCAAGUGAGCUGAAG 2578 2123UCAGCUCACUUGGCUCUCU 2579 AGAGAGCCAAGUGAGCUGA 2580 2124CAGCUCACUUGGCUCUCUA 2581 UAGAGAGCCAAGUGAGCUG 2582 2132UUGGCUCUCUAGAUCCACU 2583 AGUGGAUCUAGAGAGCCAA 2584 2134GGCUCUCUAGAUCCACUGU 2585 ACAGUGGAUCUAGAGAGCC 2586 2137UCUCUAGAUCCACUGUGGU 2587 ACCACAGUGGAUCUAGAGA 2588 2142AGAUCCACUGUGGUUGGCA 2589 UGCCAACCACAGUGGAUCU 2590 2144AUCCACUGUGGUUGGCAGU 2591 ACUGCCAACCACAGUGGAU 2592 2145UCCACUGUGGUUGGCAGUA 2593 UACUGCCAACCACAGUGGA 2594 2146CCACUGUGGUUGGCAGUAU 2595 AUACUGCCAACCACAGUGG 2596 2155UUGGCAGUAUGACCAGAAU 2597 AUUCUGGUCAUACUGCCAA 2598 2157GGCAGUAUGACCAGAAUCA 2599 UGAUUCUGGUCAUACUGCC 2600 2161GUAUGACCAGAAUCAUGGA 2601 UCCAUGAUUCUGGUCAUAC 2602 2171AAUCAUGGAAUUUGCUAGA 2603 UCUAGCAAAUUCCAUGAUU 2604 2172AUCAUGGAAUUUGCUAGAA 2605 UUCUAGCAAAUUCCAUGAU 2606 2176UGGAAUUUGCUAGAACUGU 2607 ACAGUUCUAGCAAAUUCCA 2608 2180AUUUGCUAGAACUGUGGAA 2609 UUCCACAGUUCUAGCAAAU 2610 2184GCUAGAACUGUGGAAGCUU 2611 AAGCUUCCACAGUUCUAGC 2612 2198AGCUUCUACUCCUGCAGUA 2613 UACUGCAGGAGUAGAAGCU 2614 2199GCUUCUACUCCUGCAGUAA 2615 UUACUGCAGGAGUAGAAGC 2616 2206CUCCUGCAGUAAGCACAGA 2617 UCUGUGCUUACUGCAGGAG 2618 2217AGCACAGAUCGCACUGCCU 2619 AGGCAGUGCGAUCUGUGCU 2620 2220ACAGAUCGCACUGCCUCAA 2621 UUGAGGCAGUGCGAUCUGU 2622 2221CAGAUCGCACUGCCUCAAU 2623 AUUGAGGCAGUGCGAUCUG 2624 2222AGAUCGCACUGCCUCAAUA 2625 UAUUGAGGCAGUGCGAUCU 2626 2223GAUCGCACUGCCUCAAUAA 2627 UUAUUGAGGCAGUGCGAUC 2628 2229ACUGCCUCAAUAACUUGGU 2629 ACCAAGUUAUUGAGGCAGU 2630 2231UGCCUCAAUAACUUGGUAU 2631 AUACCAAGUUAUUGAGGCA 2632 2237AAUAACUUGGUAUUGAGCA 2633 UGCUCAAUACCAAGUUAUU 2634 2240AACUUGGUAUUGAGCACGU 2635 ACGUGCUCAAUACCAAGUU 2636 2243UUGGUAUUGAGCACGUAUU 2637 AAUACGUGCUCAAUACCAA 2638 2255ACGUAUUUUGCAAAAGCUA 2639 UAGCUUUUGCAAAAUACGU 2640 2257GUAUUUUGCAAAAGCUACU 2641 AGUAGCUUUUGCAAAAUAC 2642 2258UAUUUUGCAAAAGCUACUU 2643 AAGUAGCUUUUGCAAAAUA 2644 2259AUUUUGCAAAAGCUACUUU 2645 AAAGUAGCUUUUGCAAAAU 2646 2268AAGCUACUUUUCCUAGUUU 2647 AAACUAGGAAAAGUAGCUU 2648 2271CUACUUUUCCUAGUUUUCA 2649 UGAAAACUAGGAAAAGUAG 2650 2279CCUAGUUUUCAGUAUUACU 2651 AGUAAUACUGAAAACUAGG 2652 2280CUAGUUUUCAGUAUUACUU 2653 AAGUAAUACUGAAAACUAG 2654 2312AUCCCUUUAAUUUCUUGCU 2655 AGCAAGAAAUUAAAGGGAU 2656 2326UUGCUUGAAAAUCCCAUGA 2657 UCAUGGGAUUUUCAAGCAA 2658 2327UGCUUGAAAAUCCCAUGAA 2659 UUCAUGGGAUUUUCAAGCA 2660 2329CUUGAAAAUCCCAUGAACA 2661 UGUUCAUGGGAUUUUCAAG 2662 2343GAACAUUAAAGAGCCAGAA 2663 UUCUGGCUCUUUAAUGUUC 2664 2346CAUUAAAGAGCCAGAAAUA 2665 UAUUUCUGGCUCUUUAAUG 2666 2355GCCAGAAAUAUUUUCCUUU 2667 AAAGGAAAAUAUUUCUGGC 2668 2367UUCCUUUGUUAUGUACGGA 2669 UCCGUACAUAACAAAGGAA 2670 2368UCCUUUGUUAUGUACGGAU 2671 AUCCGUACAUAACAAAGGA 2672 2369CCUUUGUUAUGUACGGAUA 2673 UAUCCGUACAUAACAAAGG 2674 2370CUUUGUUAUGUACGGAUAU 2675 AUAUCCGUACAUAACAAAG 2676 2371UUUGUUAUGUACGGAUAUA 2677 UAUAUCCGUACAUAACAAA 2678 2372UUGUUAUGUACGGAUAUAU 2679 AUAUAUCCGUACAUAACAA 2680 2373UGUUAUGUACGGAUAUAUA 2681 UAUAUAUCCGUACAUAACA 2682 2394UAUAUAGUCUUCCAAGAUA 2683 UAUCUUGGAAGACUAUAUA 2684 2401UCUUCCAAGAUAGAAGUUU 2685 AAACUUCUAUCUUGGAAGA 2686 2404UCCAAGAUAGAAGUUUACU 2687 AGUAAACUUCUAUCUUGGA 2688 2405CCAAGAUAGAAGUUUACUU 2689 AAGUAAACUUCUAUCUUGG 2690 2448UUCCAGAUAAGACAUGUCA 2691 UGACAUGUCUUAUCUGGAA 2692 2454AUAAGACAUGUCACCAUUA 2693 UAAUGGUGACAUGUCUUAU 2694 2456AAGACAUGUCACCAUUAAU 2695 AUUAAUGGUGACAUGUCUU 2696 2459ACAUGUCACCAUUAAUUCU 2697 AGAAUUAAUGGUGACAUGU 2698 2461AUGUCACCAUUAAUUCUCA 2699 UGAGAAUUAAUGGUGACAU 2700 2462UGUCACCAUUAAUUCUCAA 2701 UUGAGAAUUAAUGGUGACA 2702 2465CACCAUUAAUUCUCAACGA 2703 UCGUUGAGAAUUAAUGGUG 2704 2467CCAUUAAUUCUCAACGACU 2705 AGUCGUUGAGAAUUAAUGG 2706 2470UUAAUUCUCAACGACUGCU 2707 AGCAGUCGUUGAGAAUUAA 2708 2472AAUUCUCAACGACUGCUCU 2709 AGAGCAGUCGUUGAGAAUU 2710 2474UUCUCAACGACUGCUCUAU 2711 AUAGAGCAGUCGUUGAGAA 2712 2475UCUCAACGACUGCUCUAUU 2713 AAUAGAGCAGUCGUUGAGA 2714 2476CUCAACGACUGCUCUAUUU 2715 AAAUAGAGCAGUCGUUGAG 2716 2479AACGACUGCUCUAUUUUGU 2717 ACAAAAUAGAGCAGUCGUU 2718 2488UCUAUUUUGUUGUACGGUA 2719 UACCGUACAACAAAAUAGA 2720 2490UAUUUUGUUGUACGGUAAU 2721 AUUACCGUACAACAAAAUA 2722 2491AUUUUGUUGUACGGUAAUA 2723 UAUUACCGUACAACAAAAU 2724 2493UUUGUUGUACGGUAAUAGU 2725 ACUAUUACCGUACAACAAA 2726 2494UUGUUGUACGGUAAUAGUU 2727 AACUAUUACCGUACAACAA 2728 2495UGUUGUACGGUAAUAGUUA 2729 UAACUAUUACCGUACAACA 2730 2496GUUGUACGGUAAUAGUUAU 2731 AUAACUAUUACCGUACAAC 2732 2501ACGGUAAUAGUUAUCACCU 2733 AGGUGAUAACUAUUACCGU 2734 2506AAUAGUUAUCACCUUCUAA 2735 UUAGAAGGUGAUAACUAUU 2736 2507AUAGUUAUCACCUUCUAAA 2737 UUUAGAAGGUGAUAACUAU 2738 2521CUAAAUUACUAUGUAAUUU 2739 AAAUUACAUAGUAAUUUAG 2740 2543CACUUAUUAUGUUUAUUGU 2741 ACAAUAAACAUAAUAAGUG 2742 2555UUAUUGUCUUGUAUCCUUU 2743 AAAGGAUACAAGACAAUAA 2744 2564UGUAUCCUUUCUCUGGAGU 2745 ACUCCAGAGAAAGGAUACA 2746 2566UAUCCUUUCUCUGGAGUGU 2747 ACACUCCAGAGAAAGGAUA 2748 2571UUUCUCUGGAGUGUAAGCA 2749 UGCUUACACUCCAGAGAAA 2750 2574CUCUGGAGUGUAAGCACAA 2751 UUGUGCUUACACUCCAGAG 2752 2575UCUGGAGUGUAAGCACAAU 2753 AUUGUGCUUACACUCCAGA 2754 2580AGUGUAAGCACAAUGAAGA 2755 UCUUCAUUGUGCUUACACU 2756 2586AGCACAAUGAAGACAGGAA 2757 UUCCUGUCUUCAUUGUGCU 2758 2588CACAAUGAAGACAGGAAUU 2759 AAUUCCUGUCUUCAUUGUG 2760 2589ACAAUGAAGACAGGAAUUU 2761 AAAUUCCUGUCUUCAUUGU 2762 2594GAAGACAGGAAUUUUGUAU 2763 AUACAAAAUUCCUGUCUUC 2764 2613AUUUUUAACCAAUGCAACA 2765 UGUUGCAUUGGUUAAAAAU 2766 2619AACCAAUGCAACAUACUCU 2767 AGAGUAUGUUGCAUUGGUU 2768 2624AUGCAACAUACUCUCAGCA 2769 UGCUGAGAGUAUGUUGCAU 2770 2627CAACAUACUCUCAGCACCU 2771 AGGUGCUGAGAGUAUGUUG 2772 2628AACAUACUCUCAGCACCUA 2773 UAGGUGCUGAGAGUAUGUU 2774 2629ACAUACUCUCAGCACCUAA 2775 UUAGGUGCUGAGAGUAUGU 2776 2630CAUACUCUCAGCACCUAAA 2777 UUUAGGUGCUGAGAGUAUG 2778 2646AAAAUAGUGCCGGGAACAU 2779 AUGUUCCCGGCACUAUUUU 2780 2649AUAGUGCCGGGAACAUAGU 2781 ACUAUGUUCCCGGCACUAU 2782 2656CGGGAACAUAGUAAGGGCU 2783 AGCCCUUACUAUGUUCCCG 2784 2660AACAUAGUAAGGGCUCAGU 2785 ACUGAGCCCUUACUAUGUU 2786 2667UAAGGGCUCAGUAAAUACU 2787 AGUAUUUACUGAGCCCUUA 2788 2668AAGGGCUCAGUAAAUACUU 2789 AAGUAUUUACUGAGCCCUU 2790 2682UACUUGUUGAAUAAACUCA 2791 UGAGUUUAUUCAACAAGUA 2792 2684CUUGUUGAAUAAACUCAGU 2793 ACUGAGUUUAUUCAACAAG 2794 2698UCAGUCUCCUACAUUAGCA 2795 UGCUAAUGUAGGAGACUGA 2796 2700AGUCUCCUACAUUAGCAUU 2797 AAUGCUAAUGUAGGAGACU 2798 2702UCUCCUACAUUAGCAUUCU 2799 AGAAUGCUAAUGUAGGAGA 2800 2703CUCCUACAUUAGCAUUCUA 2801 UAGAAUGCUAAUGUAGGAG 2802 2704UCCUACAUUAGCAUUCUAA 2803 UUAGAAUGCUAAUGUAGGA 2804

Example 8 In Vivo Dose Response of EGLN Cocktail in Liver

In order to evaluate the efficacy of the iRNA agents directed to EGLNgenes, dose response studies were conducted targeting individual EGLNgenes and combinations of EGLN genes in the liver. For these studies,mice (3 animals per group) were injected IV with formulations at thedoses outlined in Table 7. A mix of EGLN1 and EGLN3, EGLN1 and EGLN2,EGLN2 and EGLN3 and EGLN1, EGLN2 and EGLN3 formulations were tested toconfirm if co-injection of individual LNP11 formulations with siRNAagainst single targets worked as well as injection of a singleformulation with siRNAs against all 3 EGLN targets. At 6 days after thesecond dose the animals were sacrificed and the livers were evaluatedfor bDNA. Serum was evaluated for EPO measurements by ELISA. The resultsare shown in FIG. 11.

It was found that each EGLN specific siRNA produced specific and robustknockdown in the liver. Furthermore, synergies were detected when thesiRNA to more than one EGLN targeting siRNA was used.

TABLE 7 In vivo knockdown of EGLN genes Dose Group siRNA (mg/kg) PBS —Luciferase AD-1955 0.5 EGLN1 AD-40894 0.5 EGLN2 AD-40773 0.5 EGLN3AD-40758 0.5 EGLN1 + 2 AD-40894 (50%) 0.5/0.5 AD-40773 (50%) EGLN1 + 3AD-40894 (50%) 0.5/0.5 AD-40758 (50%) EGLN2 + 3 AD-40773 (50%) 0.5/0.5AD-40758 (50%) EGLN1 + 2 + 3 AD-40894 (33%) 0.5/0.5/0.5 AD-40773 (33%)AD-40758 (33%)

Example 9 In Vivo Production of Erythropoietin and Hematology Using EGLNCocktail

In order to determine whether the administration of an EGLN iRNAcocktail was capable of increasing erythropoietin expression in vivo, astudy was designed according to Table 8. Female C57B6 mice were dosed IVwith PBS or LNP11-1955 luciferase controls, three different EGLN siRNAformulations or four different mixes of EGLN siRNA formulations. At day6, a second dose was administered. On day 12, plasma samples were taken,animals were sacrificed and livers were harvested for measurement ofEGLN1, EGLN2, EGLN3 and EPO mRNA. Also on day 12 blood was drawn(hematogology measurements including a count of reticulocytes, red bloodcells, hemoglobin measurements and hematocrit levels) and animals weresacrified and the livers were taken for bDNA analysis. The data areshown in FIGS. 12 and 13.

TABLE 8 In vivo knockdown of EGLN genes Dose Group siRNA (mg/kg) PBS —Luciferase AD-1955 0.5 EGLN1 AD-40894 0.5 EGLN2 AD-40773 0.5 EGLN3AD-40758 0.5 EGLN1 + 2 AD-40894 (50%) 0.5/0.5 AD-40773 (50%) EGLN1 + 3AD-40894 (50%) 0.5/0.5 AD-40758 (50%) EGLN2 + 3 AD-40773 (50%) 0.5/0.5AD-40758 (50%) EGLN1 + 2 + 3 AD-40894 (33%) 0.5/0.5/0.5 AD-40773 (33%)AD-40758 (33%)

It can be seen from FIGS. 12 and 13 that targeting EGLN1 alone or incombination with other EGLN genes increases serum EPO levels. It issuggested herein that knockdown of EGLN1 and/or EGLN2 induces feedbackloop up-regulation of EGLN3 mimicking hypoxic response.

In general, a considerable increase in reticulocytes versus control wasobsevered with an even larger increase in hematocrit, RBC count andhemoglobin levels in the plasma. Therefore, it has been surprisinglydiscovered that knockdown of EGLN1 (either alone or in combination)which produced an increase in EPO, concomitantly stimulatederythropiesis.

Example 10 Downregulation of Hepcidin

In order to evaluate the efficacy of the iRNA agents on thedownregulation of Hepcidin dose response studies were conducted foriRNAs targeting individual EGLN genes and combinations of EGLNs in theliver. For these studies, mice (5 animals per group) were injected IVwith formulations at the doses outlined in Table 9. Animals were dosedat day 1 and day 6. At day 12, the animals were bled and sacrificed andthe livers were taken. The levels of hepcidin in liver were measured bybDNA. Downregulation of Hepcidin was observed when the formulationsincluded at least EGLN1 (alone or in combination). The results are shownin FIG. 14.

TABLE 9 Downregulation of Hepcidin Dose Group siRNA (mg/kg) PBS —Luciferase AD-1955 0.5 EGLN1 AD-40894 0.5 EGLN2 AD-40773 0.5 EGLN3AD-40758 0.5 EGLN1 + 2 AD-40894 (50%) 0.5/0.5 AD-40773 (50%) EGLN1 + 3AD-40894 (50%) 0.5/0.5 AD-40758 (50%) EGLN2 + 3 AD-40773 (50%) 0.5/0.5AD-40758 (50%) EGLN1 + 2 + 3 AD-40894 (33%) 0.5/0.5/0.5 AD-40773 (33%)AD-40758 (33%)

Example 11 Tissue Specificity

In order to determine whether administration of an EGLN iRNA cocktailwas capable of tissue specificity, a study was designed according toTable 10. Female C57B6 mice were dosed four times, at day 1, 8, 15 and22, by IV with LNP11-1955 luciferase control or a cocktail of EGLN siRNAformulation. On day 29, a set of plasma samples were taken, animals weresacrificed and livers, kidneys and spleens were harvested formeasurement of EGLN1, EGLN2, EGLN3 and EPO mRNA measurements again bybranched DNA analysis.

TABLE 10 Tissue specificity Dose Group siRNA (mg/kg) Luciferase AD-1955EGLN mix AD-40894 (.375 mg/kg) 1.5 total AD-40773 (.75 mg/kg) AD-40758(.375 mg/kg)

It can be seen from FIG. 15 that the EGLN cocktail stimulated EPO in theliver and showed little to no stimulation in the kidneys and spleen.Hence the increase in serum EPO must arise from the liver. Liver tissuewas removed and stained with oil red oil and H&E and compared to thepositive control for fatty liver. Tissue staining revealed that weeklydosing (up to one month) was well tolerated by the liver.

Example 12 Durable Effects of Cocktail Administration

In order to determine durability of administration of an EGLN iRNAcocktail on the regulation of EPO and hematocrit, a study was designedaccording to Table 11. Female C57B6 mice were dosed IV with LNP11-1955luciferase control or a formulation of a mix of EGLN siRNA as outlinedin Table 11. Two groups of mice were dosed at either (1) only day 1 or(2) on days 1 and 6. At days 6, 11, 16, and 22 serum EPO was measured.At days 6, 11, 16, and 22, 27 and 33 hematocrit was measured. Theresults are shown in FIG. 16.

TABLE 11 Durable effects of cocktail administration on Epo andhematocrit Dose Group siRNA (mg/kg) Luciferase AD-1955 1.5 EGLN mixAD-40894 (.5 mg/kg) 1.5 (day 1 dose) AD-40773 (.5 mg/kg) AD-40758 (.5mg/kg) EGLN mix AD-40894 (.5 mg/kg) 1.5 (day 1 and 6 AD-40773 (.5 mg/kg)dose) AD-40758 (.5 mg/kg)

It can be seen from FIG. 16 that the knockdown by the EGLN mix wassustained over a prolonged period of time. The durability of a singledose could be seen in the samples taken for hematocrit showing lastingeffects of over one month. Prolonged effects of the administration ofthe EGLN cocktail were also seen in the increased levels of EPO whichlasted about 2 weeks after a single dose of the cocktail.

Example 13 Studies in an Animal Model of Anemia

Studies of the effects of the iRNA agents (alone or in combination) on amouse model of anemia were performed to evaluate therapeutic outcomesand efficacy. Endpoints included target knockdown of each of the EGLNgenes as well as hepcidin, improved EPO production, hematologymeasurements (including red blood cell count, Hemoglobin levels,hematocrit levels, and reticulocyte levels), and iron parameters(including serum iron level, transferrin saturation (% TSAT),unsaturated iron binding capacity (UIBC), total iron binding capacity(TIBC) and ferritin levels).

FBVN mice which had undergone 5/6 nephrectomy (Charles RiverLaboratories; Wilmington, Mass.) were dosed three times, at day 0, 4 and8. Dosing involved IV administration at 1 mg/kg of the siRNA or siRNAsoutlined in Table 12 formulated in LNP11. The study also includedcontrol groups of SHAM and PBS treated control groups as well as acontrol group containing the Luciferase siRNA AD-1955. At day 12 theanimals were sacrificed, with terminal bleeds made and tissues removedfor mRNA analysis. In all cases, the levels are normalized to levels ofactin and presented as a percent sham. The results are presented inFIGS. 17-22 and discussed below.

TABLE 12 In vivo studies in a model of anemia Sample Dose Group siRNAFormulation Size (n) (mg/kg) SHAM — 5 PBS — 5 Luciferase AD-1955 LNP11 51 (control) EGLN1 AD-40894 LNP11 4 1 EGLN1-2 AD-40894 (50%) LNP11 5 1(0.5 ea) AD-40773 (50%) EGLN1-2-3 AD-40894 (33%) LNP11 4 1 (0.33 ea) mixAD-40773 (33%) AD-40758 (33%)Target mRNA Knockdown (EGLN and Hepcidin)

Results of measurement of EGLN 1, 2, and 3 in liver as well as hepcidinexpression is shown in FIGS. 17 and 18, respectively. It can be seenfrom the data that, just as with previous studies, the effects of theiRNA agents targeting the EGLN genes, either alone or in combination arespecific and robust. There was upregulation of EGLN3 mRNA seenpreviously due to feedback regulation particularly in EGLN1-2 treatedgroups.

Downregulation of hepcidin (HAMP1) was observed when the formulationsincluded at least EGLN1 (alone or in combination). Clearly, knockdown ofEGLN1, EGLN1-2 and EGLN1-2-3 was shown to induce a down regulation ofHepcidin mRNA in the liver.

Improved EPO Production

Measurements of erythropoietin were made at the terminal bleed at day 12and the data are shown in FIG. 19. It can be seen that knockdown ofEGLN1-2 and EGLN1-2-3 significantly increased liver EPO mRNA in thecontext of 5/6 nephrectomy. An increase in EPO mRNA was not detectedwith EGLN1 knockdown consistent with previous experiments where theincrease was only seen at the protein level. These results suggest thatin anemic patients, administration of the iRNA agents targeting EGLNgenes may serve a therapeutic need to increase EPO.

Hematology

Hematocrit levels of the test groups were measured at day 0 and atsacrifice on day 12. The pre and post values of the animals are shown inFIG. 20. As can be seen from the data, there was a significant increasein Hematocrit in double and triple combo groups with a more minor effectseen in EGLN1 alone treated animals compared to SHAM controls.

Measurements of red blood cell count, Hemoglobin, and reticulocytelevels were also made at day 12 and good increases in hemoglobin andreticulocytes in all EGLN groups was observed. See FIG. 21.

Iron Parameters

Parameters associated with the etiology of anemia and erythropoiesiswere also measured at day 12. These data are presented in FIG. 22.Decreases seen in TSAT, and increases in UIBC and TIBC in the double andtriple combo EGLN knockdown animals was very informative. These datasuggest that there might not be sufficient iron available to meet theenhanced erythropoiesis demand (due to stimulation by the iRNA agentsadministered) of the system. In other words, the effect of the iRNAagents in enhancing erythropoiesis was so successful, it outpaced (ordrained) the iron pool of the animal. These data suggest that the iRNAagents may be even more effective if administered in conjunction with aniron supplement.

Example 14 Design and Synthesis of siRNA Targeting Human EGLN Genes

Oligonucleotide design was carried out to identify siRNAs targeting thegenes encoding the human (Homo sapiens) EGLN 1, 2 and 3 genes. Thedesign process used the EGLN transcript NM_(—)022051.2 for EGLN1 (SEQ IDNO: 390), NM_(—)053046.2 for EGLN2 (SEQ ID NO: 391), and NM_(—)022073.3for EGLN3 (SEQ ID NO: 392). All sequences were obtained from the NCBIRefseq collection. All siRNA duplexes were designed that shared 100%identity with the listed human and rhesus transcripts. The constructsare shown in Tables 13A, B and C.

TABLE 13A  Human EGNL1 Single Strands and Duplex SequencesFor all the sequences in the list, ‘endolight’ chemistry wasapplied as described above. SEQ SEQ Duplex Sequence (5′ to 3′) IDSequence (5′ to 3′) ID Number Sense NO Antisense NO AD-cAcGAcAccGGGAAGuucAdTsdT 2807 UGAACUUCCCGGUGUCGUGdTsdT 2808 47677.1 AD-GAcuGGGAuGccAAGGuAAdTsdT 2809 UuACCUUGGcAUCCcAGUCdTsdT 2810 47683.1 AD-ccAAGGuAAGuGGAGGuAudTsdT 2811 AuACCUCcACUuACCUUGGdTsdT 2812 47688.1 AD-GuGGAGGuAuAcuucGAAudTsdT 2813 AUUCGAAGuAuACCUCcACdTsdT 2814 47694.1 AD-GuGGAGGuAuAcuucGAAudTsdT 2815 AUUCGAAGuAuACCUCcACdTsdT 2816 47694.2 AD-GAGGuAuAcuucGAAuuuudTsdT 2817 AAAAUUCGAAGuAuACCUCdTsdT 2818 47700.1 AD-ccAAAuuuGAuAGAcuGcudTsdT 2819 AGcAGUCuAUcAAAUUUGGdTsdT 2820 47706.1 AD-GcuAcAAGGuAcGcAAuAAdTsdT 2821 UuAUUGCGuACCUUGuAGCdTsdT 2822 47711.1 AD-GAGAGcAcGAGcuAAAGuAdTsdT 2823 uACUUuAGCUCGUGCUCUCdTsdT 2824 47716.1 AD-GAGcuAAAGuAAAAuAucudTsdT 2825 AGAuAUUUuACUUuAGCUCdTsdT 2826 47678.1 AD-GuGuGAGGGuuGAAcucAAdTsdT 2827 UUGAGUUcAACCCUcAcACdTsdT 2828 47689.1 AD-GuGAGGGuuGAAcucAAuAdTsdT 2829 uAUUGAGUUcAACCCUcACdTsdT 2830 47695.1 AD-GGuuGAAcucAAuAAAccudTsdT 2831 AGGUUuAUUGAGUUcAACCdTsdT 2832 47701.1 AD-GAcGucuucuAGAGccuuudTsdT 2833 AAAGGCUCuAGAAGACGUCdTsdT 2834 47707.1 AD-ccAGAucuGuuAucuAGcudTsdT 2835 AGCuAGAuAAcAGAUCUGGdTsdT 2836 47712.1 AD-GuuAucuAGcuGAGuucAudTsdT 2837 AUGAACUcAGCuAGAuAACdTsdT 2838 47717.1 AD-GGuAcAAuuuAucuAAAcudTsdT 2839 AGUUuAGAuAAAUUGuACCdTsdT 2840 47679.1 AD-ccucuuAAuAAuGAuuGuudTsdT 2841 AAcAAUcAUuAUuAAGAGGdTsdT 2842 47684.1 AD-GccAGuGAcuGAuGAuuAAdTsdT 2843 UuAAUcAUcAGUcACUGGCdTsdT 2844 47690.1 AD-ccAGuGAcuGAuGAuuAAudTsdT 2845 AUuAAUcAUcAGUcACUGGdTsdT 2846 47696.1 AD-GAGcAcuuuAAuuAcAAcudTsdT 2847 AGUUGuAAUuAAAGUGCUCdTsdT 2848 47702.1 AD-ccAuuuAcuAccAAuAAcudTsdT 2849 AGUuAUUGGuAGuAAAUGGdTsdT 2850 47708.1 AD-GGcuGGGGuuuAAGuuAAAdTsdT 2851 UUuAACUuAAACCCcAGCCdTsdT 2852 47713.1 AD-GcuGGGGuuuAAGuuAAAudTsdT 2853 AUUuAACUuAAACCCcAGCdTsdT 2854 47718.1 AD-cuucAAGuuccuAAGAuAAdTsdT 2855 UuAUCUuAGGAACUUGAAGdTsdT 2856 47680.1 AD-GGGcuuucuuAAGcuuucAdTsdT 2857 UGAAAGCUuAAGAAAGCCCdTsdT 2858 47685.1 AD-cuuAGAcuucAcuuuccuAdTsdT 2859 uAGGAAAGUGAAGUCuAAGdTsdT 2860 47691.1 AD-cuucAcuuuccuAGGcuuudTsdT 2861 AAAGCCuAGGAAAGUGAAGdTsdT 2862 47697.1 AD-cuAucucuGuccuuGAucudTsdT 2863 AGAUcAAGGAcAGAGAuAGdTsdT 2864 47703.1 AD-GccAAAAuGuGAGuAuAcAdTsdT 2865 UGuAuACUcAcAUUUUGGCdTsdT 2866 47709.1 AD-cAAAAuGuGAGuAuAcAGAdTsdT 2867 UCUGuAuACUcAcAUUUUGdTsdT 2868 47714.1 AD-cuuAGGAGAAuuuGcAGGAdTsdT 2869 UCCUGcAAAUUCUCCuAAGdTsdT 2870 47719.1 AD-GcGuuAGGccAcAAcucAAdTsdT 2871 UUGAGUUGUGGCCuAACGCdTsdT 2872 47686.1 AD-cGuuAGGccAcAAcucAAAdTsdT 2873 UUUGAGUUGUGGCCuAACGdTsdT 2874 47692.1 AD-cuAucuGuGGGuuGuGcuudTsdT 2875 AAGcAcAACCcAcAGAuAGdTsdT 2876 47698.1 AD-cAGAcAGGucuuAAAuuGudTsdT 2877 AcAAUUuAAGACCUGUCUGdTsdT 2878 47704.1 AD-GGAAAAGuuuAuAuAcucudTsdT 2879 AGAGuAuAuAAACUUUUCCdTsdT 2880 47710.1 AD-cuGuuuGuGGccuAuAuGudTsdT 2881 AcAuAuAGGCcAcAAAcAGdTsdT 2882 47715.1 AD-GuuuGuGGccuAuAuGuGudTsdT 2883 AcAcAuAuAGGCcAcAAACdTsdT 2884 47720.1 AD-GuGuuuAAuccuGGuuAAAdTsdT 2885 UUuAACcAGGAUuAAAcACdTsdT 2886 47682.1 AD-GuuuAAuccuGGuuAAAGAdTsdT 2887 UCUUuAACcAGGAUuAAACdTsdT 2888 47687.1 AD-cuGuuuuuAuucAAcAcAudTsdT 2889 AUGUGUUGAAuAAAAAcAGdTsdT 2890 47693.1 AD-cAuAuAcAGAuAGAcuAuAdTsdT 2891 uAuAGUCuAUCUGuAuAUGdTsdT 2892 47699.1 AD-cAAGuuGcuuGuAAAGcuAdTsdT 2893 uAGCUUuAcAAGcAACUUGdTsdT 2894 47705.1 AD-GcuuGuAAAGcuAAucuAAdTsdT 2895 UuAGAUuAGCUUuAcAAGCdTsdT 2896 40932.2 AD-GcuuGuAAAGcuAAucuAAdTsdT 2897 UuAGAUuAGCUUuAcAAGCdTsdT 2898 40932.1 AD-GcuuGuAAAGcuAAucuAAdTsdT 2899 UuAGAUuAGCUUuAcAAGCdTsdT 2900 40932.3

TABLE 13B  Human EGNL2 Single Strands and Duplex SequencesFor all the sequences in the list, ‘endolight’ chemistrywas applied as described above. SEQ SEQ Duplex Sequence (5′ to 3′) IDSequence (5′ to 3′) ID Number Sense NO Antisense NO AD-cuucccAAGcccuuAGGGAdTsdT 2901 UCCCuAAGGGCUUGGGAAGdTsdT 2902 47721.1 AD-cuuGGGGAccAGcAAGcAAdTsdT 2903 UUGCUUGCUGGUCCCcAAGdTsdT 2904 47727.1 AD-cAuGcccGGGGGAuGAAGAdTsdT 2905 UCUUcAUCCCCCGGGcAUGdTsdT 2906 47733.1 AD-cccGGGGGAuGAAGAcAcudTsdT 2907 AGUGUCUUcAUCCCCCGGGdTsdT 2908 47738.1 AD-GGGGGAuGAAGAcAcuGcudTsdT 2909 AGcAGUGUCUUcAUCCCCCdTsdT 2910 47744.1 AD-GcAGccccuAAGucAGGcudTsdT 2911 AGCCUGACUuAGGGGCUGCdTsdT 2912 47750.1 AD-cAGuuAccAGGGucuucGudTsdT 2913 ACGAAGACCCUGGuAACUGdTsdT 2914 47756.1 AD-GAGGcccccAAAcGGAAAudTsdT 2915 AUUUCCGUUUGGGGGCCUCdTsdT 2916 47722.1 AD-GGGccAGGcAAGAGAAccAdTsdT 2917 UGGUUCUCUUGCCUGGCCCdTsdT 2918 47728.1 AD-GccuGGcccuGGAcuAuAudTsdT 2919 AuAuAGUCcAGGGCcAGGCdTsdT 2920 47734.1 AD-GcAuGcGGuAcuAcGGcAudTsdT 2921 AUGCCGuAGuACCGcAUGCdTsdT 2922 47739.1 AD-GGuAcuAcGGcAucuGcGudTsdT 2923 ACGcAGAUGCCGuAGuACCdTsdT 2924 47745.1 AD-cAuccGuGGGGAccAGAuudTsdT 2925 AAUCUGGUCCCcACGGAUGdTsdT 2926 47751.1 AD-cGGGuAcGuAAGGcAcGuudTsdT 2927 AACGUGCCUuACGuACCCGdTsdT 2928 47763.1 AD-GGuAcGuAAGGcAcGuuGAdTsdT 2929 UcAACGUGCCUuACGuACCdTsdT 2930 47723.1 AD-cGcuGcAucAccuGuAucudTsdT 2931 AGAuAcAGGUGAUGcAGCGdTsdT 2932 47729.1 AD-GcAucAccuGuAucuAuuAdTsdT 2933 uAAuAGAuAcAGGUGAUGCdTsdT 2934 40743.2 AD-GcAucAccuGuAucuAuuAdTsdT 2935 uAAuAGAuAcAGGUGAUGCdTsdT 2936 40743.1 AD-ccuGuAucuAuuAccuGAAdTsdT 2937 UUcAGGuAAuAGAuAcAGGdTsdT 2938 47740.1 AD-GuAucuAuuAccuGAAucAdTsdT 2939 UGAUUcAGGuAAuAGAuACdTsdT 2940 47746.1 AD-GAAucAGAAcuGGGAcGuudTsdT 2941 AACGUCCcAGUUCUGAUUCdTsdT 2942 47752.1 AD-cuGGGAcGuuAAGGuGcAudTsdT 2943 AUGcACCUuAACGUCCcAGdTsdT 2944 47758.1 AD-cucuuuGAccGGuuGcucAdTsdT 2945 UGAGcAACCGGUcAAAGAGdTsdT 2946 47764.1 AD-cuuuGAccGGuuGcucAuudTsdT 2947 AAUGAGcAACCGGUcAAAGdTsdT 2948 47724.1 AD-GAccGGuuGcucAuuuucudTsdT 2949 AGAAAAUGAGcAACCGGUCdTsdT 2950 47730.1 AD-GuGAAGccAGccuAuGccAdTsdT 2951 UGGcAuAGGCUGGCUUcACdTsdT 2952 47735.1 AD-ccAGGuAcGccAucAcuGudTsdT 2953 AcAGUGAUGGCGuACCUGGdTsdT 2954 47741.1 AD-ccAucAcuGucuGGuAuuudTsdT 2955 AAAuACcAGAcAGUGAUGGdTsdT 2956 47747.1 AD-GcAGcAGccAAAGAcAAGudTsdT 2957 ACUUGUCUUUGGCUGCUGCdTsdT 2958 47753.1 AD-cAGccAAAGAcAAGuAucAdTsdT 2959 UGAuACUUGUCUUUGGCUGdTsdT 2960 47759.1 AD-cAGccAAAGAcAAGuAucAdTsdT 2961 UGAuACUUGUCUUUGGCUGdTsdT 2962 47759.2 AD-cAAAGAcAAGuAucAGcuAdTsdT 2963 uAGCUGAuACUUGUCUUUGdTsdT 2964 47765.1 AD-GAcAAGuAucAGcuAGcAudTsdT 2965 AUGCuAGCUGAuACUUGUCdTsdT 2966 47725.1 AD-GuAucAGcuAGcAucAGGAdTsdT 2967 UCCUGAUGCuAGCUGAuACdTsdT 2968 47731.1 AD-cAGcuAGcAucAGGAcAGAdTsdT 2969 UCUGUCCUGAUGCuAGCUGdTsdT 2970 47736.1 AD-GcuAGcAucAGGAcAGAAAdTsdT 2971 UUUCUGUCCUGAUGCuAGCdTsdT 2972 47742.1 AD-GAAAGGuGuccAAGuAccudTsdT 2973 AGGuACUUGGAcACCUUUCdTsdT 2974 47748.1 AD-ccuAGuGGccAGucccAGAdTsdT 2975 UCUGGGACUGGCcACuAGGdTsdT 2976 47754.1 AD-cuGucuGGucAuGAccccAdTsdT 2977 UGGGGUcAUGACcAGAcAGdTsdT 2978 47760.1 AD-GucuGGucAuGAccccAuudTsdT 2979 AAUGGGGUcAUGACcAGACdTsdT 2980 47766.1 AD-cuGGGAGGAGGcAuuGucAdTsdT 2981 UGAcAAUGCCUCCUCCcAGdTsdT 2982 47726.1 AD-GGAGGAGGcAuuGucAcuudTsdT 2983 AAGUGAcAAUGCCUCCUCCdTsdT 2984 47732.1 AD-GcAuuGucAcuucccAccAdTsdT 2985 UGGUGGGAAGUGAcAAUGCdTsdT 2986 47737.1 AD-GGAcuuGGGGuuGAGGuGAdTsdT 2987 UcACCUcAACCCcAAGUCCdTsdT 2988 47743.1 AD-cucuuGcuGGcAAuGGGGudTsdT 2989 ACCCcAUUGCcAGcAAGAGdTsdT 2990 47749.1 AD-ccAGccuGGAAuGuGAAGudTsdT 2991 ACUUcAcAUUCcAGGCUGGdTsdT 2992 47755.1 AD-GGcAGAGuAAAAGGuGccAdTsdT 2993 UGGcACCUUUuACUCUGCCdTsdT 2994 47761.1

TABLE 13C  Human EGNL3 Single Strands and Duplex SequencesFor all the sequences in the list, ‘endolight’ chemistry wasapplied as described above. SEQ SEQ Duplex Sequence (5′ to 3′) IDSequence (5′ to 3′) ID Number Sense NO Antisense NO AD-GuGGcAGccGcAGGuuucudTsdT 2995 AGAAACCUGCGGCUGCcACdTsdT 2996 47767.1 AD-GcAGccGcAGGuuucuGAAdTsdT 2997 UUcAGAAACCUGCGGCUGCdTsdT 2998 47773.1 AD-GGcuucGcGcucGuGuAGAdTsdT 2999 UCuAcACGAGCGCGAAGCCdTsdT 3000 47779.1 AD-GcuucGcGcucGuGuAGAudTsdT 3001 AUCuAcACGAGCGCGAAGCdTsdT 3002 47785.1 AD-cGcGcucGuGuAGAucGuudTsdT 3003 AACGAUCuAcACGAGCGCGdTsdT 3004 47791.1 AD-GAucccGGAccucGAuucudTsdT 3005 AGAAUCGAGGUCCGGGAUCdTsdT 3006 47797.1 AD-cAAGGAGAGGucuAAGGcAdTsdT 3007 UGCCUuAGACCUCUCCUUGdTsdT 3008 47803.1 AD-GGcAAuGGuGGcuuGcuAudTsdT 3009 AuAGcAAGCcACcAUUGCCdTsdT 3010 47809.1 AD-ccGGGAAAuGGAAcAGGuudTsdT 3011 AACCUGUUCcAUUUCCCGGdTsdT 3012 47768.1 AD-ccuGcAucuAcuAucuGAAdTsdT 3013 UUcAGAuAGuAGAUGcAGGdTsdT 3014 47786.1 AD-GAuccuGcGGAuAuuuccAdTsdT 3015 UGGAAAuAUCCGcAGGAUCdTsdT 3016 47792.1 AD-GGGGAAAucAuucAuAGcAdTsdT 3017 UGCuAUGAAUGAUUUCCCCdTsdT 3018 47798.1 AD-GGAAAucAuucAuAGcAGAdTsdT 3019 UCUGCuAUGAAUGAUUUCCdTsdT 3020 47804.1 AD-GAcAGAcuccuGuucuucudTsdT 3021 AGAAGAAcAGGAGUCUGUCdTsdT 3022 47769.1 AD-ccuGuucuucuGGucAGAudTsdT 3023 AUCUGACcAGAAGAAcAGGdTsdT 3024 47775.1 AD-GcAAccAGAuAuGcuAuGAdTsdT 3025 UcAuAGcAuAUCUGGUUGCdTsdT 3026 47781.1 AD-ccAGAuAuGcuAuGAcuGudTsdT 3027 AcAGUcAuAGcAuAUCUGGdTsdT 3028 47787.1 AD-cuAuGAcuGucuGGuAcuudTsdT 3029 AAGuACcAGAcAGUcAuAGdTsdT 3030 47793.1 AD-GAAAuucAGGAAuuuAAcudTsdT 3031 AGUuAAAUUCCUGAAUUUCdTsdT 3032 47805.1 AD-GAAuuuAAcuAGGAAAAcudTsdT 3033 AGUUUUCCuAGUuAAAUUCdTsdT 3034 47811.1 AD-GccuuGuucAuuuuAGuAAdTsdT 3035 UuACuAAAAUGAAcAAGGCdTsdT 3036 47770.1 AD-GuuccuGAAuucucuuAAAdTsdT 3037 UUuAAGAGAAUUcAGGAACdTsdT 3038 47776.1 AD-GuuccuGAAuucucuuAAAdTsdT 3039 UUuAAGAGAAUUcAGGAACdTsdT 3040 47776.2 AD-cuGAAuucucuuAAAuucudTsdT 3041 AGAAUUuAAGAGAAUUcAGdTsdT 3042 47782.1 AD-cAAAGAuGGccucuucAGudTsdT 3043 ACUGAAGAGGCcAUCUUUGdTsdT 3044 47788.1 AD-cuGcuAcuucuuGcAuccudTsdT 3045 AGGAUGcAAGAAGuAGcAGdTsdT 3046 47800.1 AD-cccuGucuuGuGuGuGGuAdTsdT 3047 uACcAcAcAcAAGAcAGGGdTsdT 3048 47806.1 AD-cuuGuGuGuGGuAcuucAudTsdT 3049 AUGAAGuACcAcAcAcAAGdTsdT 3050 47812.1 AD-GuGuGGuAcuucAuGuuuudTsdT 3051 AAAAcAUGAAGuACcAcACdTsdT 3052 47771.1 AD-GuuuucuuGccAAGAcuGudTsdT 3053 AcAGUCUUGGcAAGAAAACdTsdT 3054 47777.1 AD-cGAGGGAAuGAAccuuAcudTsdT 3055 AGuAAGGUUcAUUCCCUCGdTsdT 3056 47783.1 AD-cuuAcuuGcAcuuuAuGuAdTsdT 3057 uAcAuAAAGUGcAAGuAAGdTsdT 3058 47789.1 AD-cAcuuuAuGuAuAcuuccudTsdT 3059 AGGAAGuAuAcAuAAAGUGdTsdT 3060 47795.1 AD-GuAuAcuuccuGAuuuGAAdTsdT 3061 UUcAAAUcAGGAAGuAuACdTsdT 3062 47801.1 AD-GGAGAAuuAucAcAAccuAdTsdT 3063 uAGGUUGUGAuAAUUCUCCdTsdT 3064 47807.1 AD-ccuAAuGAcAuuAAuAccudTsdT 3065 AGGuAUuAAUGUcAUuAGGdTsdT 3066 47813.1 AD-cccuGGuAGuuuuGuGuuAdTsdT 3067 uAAcAcAAAACuACcAGGGdTsdT 3068 47772.1 AD-ccuGGuAGuuuuGuGuuAAdTsdT 3069 UuAAcAcAAAACuACcAGGdTsdT 3070 47778.1 AD-GuGGAAAGAGcuAGGucuAdTsdT 3071 uAGACCuAGCUCUUUCcACdTsdT 3072 47784.1 AD-cuAGGucuAcuGAuAuAcAdTsdT 3073 UGuAuAUcAGuAGACCuAGdTsdT 3074 47790.1 AD-GucuAcuGAuAuAcAAuAAdTsdT 3075 UuAUUGuAuAUcAGuAGACdTsdT 3076 47796.1 AD-cAuGuGuGcAucuuGAAcAdTsdT 3077 UGUUcAAGAUGcAcAcAUGdTsdT 3078 47802.1 AD-GuGuGcAucuuGAAcAAuudTsdT 3079 AAUUGUUcAAGAUGcAcACdTsdT 3080 47808.1

Example 15 Studies of siRNA in an Animal Model:Hematology Measurements

Studies of the effects of siRNA agents in combination on a mouse modelwere performed to evaluate therapeutic outcomes and efficacy. Endpointsincluded hematology measurements (including red blood cell count,Hemoglobin levels, hematocrit levels, and reticulocyte levels).

Wild type C57BL/6 mice were dosed two times, at day 0 and 6. Dosinginvolved tail vein administration of an equal part mixture of the threesiRNAs (AD-40894, AD-40773 and AD-40758) targeting EGLN1, EGLN2, andEGLN3 respectively. The study also included control groups of PBStreated control and a control group containing the luciferase siRNAAD-1955. The results are presented in Table 14.

Hematology

Hematocrit levels of the test group were measured at day 4 and 9. As canbe seen from the data in Table 14, there was an increase in hematocritin the mice treated with an equal part mixture of siRNAs as compared tothe PBS and Luciferase controls. Measurements of red blood cell count,Hemoglobin, and reticulocyte levels were also made at day 4 and 9 and anincrease in Hemoglobin and reticulocyte levels was observed. These dataare also presented in Table 14. In the table “Hg” stands for Hemoglobinin g/dL, “HCT” stands for Hematocrit in %, “Ret” stands forReticulocytes in %, and “RBC” stands for Red Blood Cells (×10⁶cells/uL).

TABLE 14 In vivo studies in an animal model Day 4 Bleed Day 9 Bleed RetRBC Hg HCT Ret RBC Hg HCT PBS 3.4 8.7 12.6 40.6 7.4 8.3 12.2 39.3Luciferase 3.2 8.6 12.3 39.7 7.1 7.7 11.3 36.2 EGLN 1, 2, 3 10.1 9.413.6 45.8 12.5 10.5 15.4 52.9

Additional Hematology Studies: Day 0 and Day 5Dosing

Studies on the effects of the siRNA agents (alone or in combination) ona mouse model were performed to evaluate the effect of the siRNA agentson EPO production and erythropoiesis. Endpoints included hematologymeasurements (including red blood cell count, Hemoglobin levels,hematocrit levels, and reticulocyte levels). Wild type C57BL/6 mice weredosed two times, at day 0 and 5. Dosing involved tail veinadministration at 0.5 mg/kg per EGLN family member, EGLN1 (AD-40894),EGLN2 (AD-40773), and EGLN3 (AD-40758). The study also included controlgroups of PBS treated mice and a group containing the luciferase siRNAAD-1955.

Hematology

Hematocrit levels of the test group were measured at sacrifice on day11. The values are shown in Table 15 along with reticulocyte levels,hemoglobin levels and red blood cell count.

TABLE 15 In vivo studies in an animal model on day 11 Red BloodReticulocyte Cell Hemoglobin Hematocrit PBS 3.7 8.5 12.8 40.5 Luciferase2.9 8.7 12.9 41.7 EGLN1 8.7 10.6 15.4 52.3 EGLN2 3.8 8.7 12.4 40.1 EGLN33.6 8.3 12.3 40.0 EGLN1, 2 10.6 11.5 16.5 56.2 EGLN2, 3 7.6 10.0 14.849.3 EGLN1, 3 4.6 8.0 12.0 39.1 EGLN 1, 2, 3 12.2 11.9 16.9 58.5

Example 16 5′RACE Assay

A 5′RACE assay was used in order to monitor the cleavage site of targetmRNA. The 5′RACE analysis showed that the downregulation of EGLN mRNA inthe liver was specifically due to siRNA-mediated mRNA cleavage. Table 16lists the 5′RACE primers used in this analysis.

TABLE 16  5′RACE Primers Sequence 5′ to 3′ SEQ ID NO. Adaptor oligoCGACTGGAGCACGAGGACACTGACATGG 3081 Nested GGACACTGACATGGACTGAAGGAGTAG3082 Adaptor oligo EGLN1 GSP AGAGATGAAATGAACTCAGTTAGGTGACAGGTCTG 3083EGLN1 PCR TTGTTTCGTGTCCAGATGGAAAAGCTACTCTCCTC 3084 Round 1 EGLN1 PCRGGCTTGAGTTCAACCCTCACACCTTTCTCACCTG 3085 Round 2 EGLN2 GSPTATTTCTTGGCTGGCAGAACCTCCATAC 3086 EGLN2 PCRCAGACAGTGGCAGCCCAGTCCATACACTG 3087 Round 1 EGLN2 PCRCAGCAGAGGTCTCTCCTTGTTGCTCCTCAGTG 3088 Round 2 EGLN3 GSPGATGTGGAAGAACTCCAATAGCTCTGAGGTC 3089 EGLN3 PCRCAGTGCTGAATTACCAGGAAGCTTTCTATCCTCTG 3090 Round 1 EGLN3 PCRGCAAGAAAACATGAAGTACCACAAACAAG 3091 Round 2

Example 17 Animal Model: Anemia

We next asked if EGLN siRNA could be used to treat anemia in the settingof chronic renal failure. Toward this end mice were subjected to 5/6nephrectomy, which is a widely used model for anemia linked to renalfailure, or sham operations (FIG. 23). The mice undergoing nephrectomydeveloped anemia, as expected, and were then randomized to receivephosphate buffered saline (PBS), control siRNA (luciferase siRNA),siRNAs targeting EglN¹, EGLN1 and EGLN2, or combinations thereof. Inkeeping with the data described above, inactivation of EGLN1 led to amodest increase in red blood cell production, which was markedlyaccentuated by coinactivation of EGLN2. Treatment with EGLN1 and EGLN2constructs restored both hemoglobin and hematocrit levels (FIG. 23 B,C). The maximal erythropoietic response, however, was observed aftertreatment with siRNA targeting all 3 EGLN paralogs. EglN inactivation inthis model also led to an upregulation of EPO and a decrease in hepcidinmRNA levels, consistent with earlier studies using chemical hydroxylaseinhibitors (FIG. 24).

Chronic inflammation can lead to anemia due, at least partly, toincreased levels of hepcidin and altered iron trafficking (anemia ofchronic disease). Rats with experimental arthritis induced by a polymerof a streptococcal antigen (PG-APS) have been used as a model for theanemia linked to inflammation (M. A. Coccia et al., Exp Hematol 29, 1201(October, 2001); R. B. Sartor et al., Infect Immun 57, 1177 (April,1989); W. J. Cromartie, J. G. Craddock, J. H. Schwab, S. K. Anderle, C.H. Yang, J Exp Med 146, 1585 (Dec. 1, 1977). In the 5/6 nephrectomymodel combined inactivation of EGLN1 and EGLN2 was sufficient to inducea brisk erythropoietic response (FIG. 23) and we were able to identifysiRNAs that can effectively target rat EglN1 and EglN2 (FIG. 25A-C).Treatment of anemic PG-APS rats with mixtures of siRNAs targeting bothEglN1 and EglN2 decreased their hepcidin levels and corrected theiranemia (FIG. 25).

These studies suggest that systemically administered siRNAs targetingthe EGLN family would ameliorate anemias characterized by an absolute orrelative deficiency of erythropoietin, such as anemias linked to chronickidney disease or inflammation, in man. This approach would allow thebody to produce native erythropoietin, thereby obviating the need forrecombinant versions of this hormone. Moreover other hepatic changesinduced by EGLN inhibition, such as decreased production of hepcidin,might enhance the effectiveness of endogenous erythropoietin and therebylower the circulating erythropoietin levels needed to promote red bloodcell production. This might be desirable if some of the cardiovascularcomplications of chronic erythropoietin production are more tightlylinked to circulating erythropoietin levels, especially whensupraphysiological, than to red blood cell mass per se.

Example 18 Decrease of Hepatic EGLN Activity: Photon Emission Study

It has previously been shown that EGLN activity can be monitorednon-invasively in mice that ubiquitously express a HIF1α-luciferasefusion protein that contains a region of HIF1α that is sufficient to behydroxylated by EGLN and subsequently ubiquitinated by the pVHLubiquitin ligase complex (M. Safran et al., Proc Nall Acad Sci USA 103,105 (Jan. 3, 2006). As expected, administration of the EGLN siRNA mix tothese mice decreased hepatic, but not renal, EGLN activity as determinedby increased photon emission in the region of the liver, but notkidneys, following luciferin administration (See FIG. 26). Branched DNAanalysis confirmed that EglN1, EglN2, and EglN3 mRNAs were decreased inthe liver, but not the kidney, and was associated with an increasehepatic, but not renal, EPO mRNA production.

It is to be understood that the words which have been used are words ofdescription rather than limitation, and that changes may be made withinthe purview of the appended claims without departing from the true scopeand spirit of the invention in its broader aspects.

While the present invention has been described at some length and withsome particularity with respect to the several described embodiments, itis not intended that it should be limited to any such particulars orembodiments or any particular embodiment, but it is to be construed withreferences to the appended claims so as to provide the broadest possibleinterpretation of such claims in view of the prior art and, therefore,to effectively encompass the intended scope of the invention.

All publications, patent applications, patents, and other referencesmentioned herein are incorporated by reference in their entirety. Incase of conflict, the present specification, including definitions, willcontrol. In addition, section headings, the materials, methods, andexamples are illustrative only and not intended to be limiting.

1. A composition comprising at least one double-stranded ribonucleicacid (dsRNA) for inhibiting expression of EGLN1, wherein said dsRNAcomprises a sense strand and an antisense strand, wherein the sensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of sequences listed inTables 2A-F and 6A-C and the antisense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of sequences listed in Tables 2A-F and 6A-C.
 2. Thecomposition of claim 1 further comprising at least one double-strandedribonucleic acid (dsRNA) for inhibiting expression of EGLN2, EGLN3 or acombination thereof wherein said dsRNA inhibiting expression of EGLN2and/or EGLN3 comprises a sense strand and an antisense strand, whereinthe sense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of sequenceslisted in Tables 2A-F and 6A-C and the antisense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of sequences listed in Tables 2A-F and6A-C.
 3. The composition of claim 2 wherein the double-strandedribonucleic acid (dsRNA) for inhibiting expression of EGLN1 comprises asense strand and an antisense strand, wherein the sense strand comprisesat least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of (SEQ ID NO: 26) and theantisense strand comprises at least 15 contiguous nucleotides differingby no more than 3 nucleotides from the nucleotide sequence of (SEQ IDNO: 27) and wherein the double-stranded ribonucleic acid (dsRNA) forinhibiting expression of EGLN3, comprises a sense strand and anantisense strand, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of (SEQ ID NO: 282) and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of (SEQ ID NO: 283).
 4. Thecomposition of claim 3 wherein the double-stranded ribonucleic acid(dsRNA) for inhibiting expression of EGLN2 comprises a sense strand andan antisense strand, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of (SEQ ID NO: 176) and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of (SEQ ID NO: 177).
 5. Thecomposition of claim 2, wherein each dsRNA targeting each of EGLN1,EGLN2 and EGLN3 comprises at least one modified nucleotide.
 6. Thecomposition of claim 5, wherein at least one of said modifiednucleotides is chosen from the group consisting of: a 2′-O-methylmodified nucleotide, a nucleotide comprising a 5′-phosphorothioategroup, and a terminal nucleotide linked to a cholesteryl derivative ordodecanoic acid bisdecylamide group.
 7. The composition of claim 6,wherein said modified nucleotide is chosen from the group consisting of:a 2′-deoxy-2′-fluoro modified nucleotide, a 2′-deoxy-modifiednucleotide, a locked nucleotide, an abasic nucleotide, 2′-amino-modifiednucleotide, 2′-alkyl-modified nucleotide, morpholino nucleotide, aphosphoramidate, and a non-natural base comprising nucleotide.
 8. Thecomposition of claim 7, wherein the region of complementarity is atleast 17 nucleotides in length.
 9. The composition of claim 8, whereinthe region of complementarity is between 19 and 21 nucleotides inlength.
 10. The composition of claim 9, wherein the region ofcomplementarity is 19 nucleotides in length.
 11. The composition ofclaim 1 or 2, wherein each strand is no more than 30 nucleotides inlength.
 12. The composition of claim 11, wherein at least one strandcomprises a 3′ overhang of at least 1 nucleotide.
 13. The composition ofclaim 12, wherein at least one strand comprises a 3′ overhang of atleast 2 nucleotides.
 14. The composition of claim 13 wherein thedouble-stranded ribonucleic acid (dsRNA) for inhibiting expression ofEGLN1 comprises a sense strand and an antisense strand, wherein thesense strand comprises at least 15 contiguous nucleotides differing byno more than 3 nucleotides from the nucleotide sequence of (SEQ ID NO:88) and the antisense strand comprises at least 15 contiguousnucleotides differing by no more than 3 nucleotides from the nucleotidesequence of (SEQ ID NO: 89) and wherein the double-stranded ribonucleicacid (dsRNA) for inhibiting expression of EGLN3, comprises a sensestrand and an antisense strand, wherein the sense strand comprises atleast 15 contiguous nucleotides differing by no more than 3 nucleotidesfrom the nucleotide sequence of (SEQ ID NO: 346) and the antisensestrand comprises at least 15 contiguous nucleotides differing by no morethan 3 nucleotides from the nucleotide sequence of (SEQ ID NO: 347). 15.The composition of claim 14 wherein the double-stranded ribonucleic acid(dsRNA) for inhibiting expression of EGLN2 comprises a sense strand andan antisense strand, wherein the sense strand comprises at least 15contiguous nucleotides differing by no more than 3 nucleotides from thenucleotide sequence of (SEQ ID NO: 240) and the antisense strandcomprises at least 15 contiguous nucleotides differing by no more than 3nucleotides from the nucleotide sequence of (SEQ ID NO: 241).
 16. Thecomposition of claim 2, further comprising a ligand.
 17. The compositionof claim 16, wherein the ligand is conjugated to the 3′ end of the sensestrand of the dsRNA.
 18. A cell containing the dsRNA of claim 1 or 2.19. A pharmaceutical composition for inhibiting expression of an EGLNgene comprising the dsRNA of claim 1 or
 2. 20. The pharmaceuticalcomposition of claim 1 or 2, further comprising a lipid formulation. 21.The pharmaceutical composition of claim 20, wherein the lipidformulation is a MC3 formulation
 22. A method of inhibiting EGLN1, EGLN2and EGLN3 expression in a cell, the method comprising: (a) introducinginto the cell the composition of claim 1 or 2; and (b) maintaining thecell produced in step (a) for a time sufficient to obtain degradation ofthe mRNA transcript of an EGLN gene, thereby inhibiting expression ofthe EGLN gene in the cell.
 23. The method of claim 22, wherein EGLN1,EGLN2 and EGLN3 expression are each inhibited by at least 30%.
 24. Amethod of treating a disorder mediated by EGLN expression comprisingadministering to a human in need of such treatment a therapeuticallyeffective amount of the composition of claim 1 or
 2. 25. The method ofclaim 24, wherein the human has anemia or a condition associated withanemia.
 26. The method of claim 25, wherein the anemia is selected fromthe group consisting of anemia due to B 12 deficiency, anemia due tofolate deficiency, anemia due to iron deficiency, hemolytic anemia,hemolytic anemia due to G-6-PD deficiency, idiopathic aplastic anemia,idiopathic autoimmune hemolytic anemia, immune hemolytic anemia,iegaloblastic anemia, pernicious anemia, secondary aplastic anemia, andsickle cell anemia.
 27. The method of claim 25, wherein the conditionassociated with anemia is selected from the group consisting of paleskin, dizziness, fatigue, headaches, irritability, low body temperature,numb/cold hands or feet, rapid heartbeat, reduced erythropoietin,shortness of breath, weakness and chest pain.
 28. The method of claim24, wherein the human has a disorder selected from the group consistingof hypoxia, a neurological condition, renal disease or failure, andcancers of the blood, bone and marrow.
 29. A method of increasingerythropoietin levels in a cell or organism comprising contacting saidcell or organism with the composition of claim 1 or
 2. 30. A method ofincreasing erythropoietin levels in a cell or organism comprisingcontacting said cell or organism with the composition of claim 3 or 4.31. A method of increasing erythropoietin levels in a cell or organismcomprising contacting said cell or organism with the composition ofclaim 14 or
 15. 32. A composition comprising at least onedouble-stranded ribonucleic acid (dsRNA) for inhibiting expression ofEGLN1, wherein said dsRNA consists of a sense strand and an antisensestrand, wherein the sense strand consists of the nucleotide sequence ofSEQ ID NO: 2807 and the antisense strand consist of the nucleotidesequence of SEQ ID NO: 2808.